Treatment and prognostic monitoring of non-cancerous proliferation disorders using hedgehog pathway inhibitors

ABSTRACT

The present invention concerns methods for treating a proliferation disorder, such as prostate cancer, basal cell carcinoma, lung cancer, and other cancers, using an inhibitor of the Hedgehog pathway (HhP); and methods for monitoring subjects undergoing such treatments based on biomarkers and other criteria predictive of efficacy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/420,283, filed Jan. 31, 2017, which is a continuation of U.S.application Ser. No. 14/947,345, filed Nov. 20, 2015, now abandoned,which is a continuation of U.S. application Ser. No. 14/173,588, filedFeb. 5, 2014, now U.S. Pat. No. 9,192,609, which claims the benefit ofU.S. Provisional Application Ser. No. 61/831,823, filed Jun. 6, 2013 andU.S. Provisional Application Ser. No. 61/813,122, filed Apr. 17, 2013,which are hereby incorporated by reference herein in their entirety,including any figures, tables, or drawings.

BACKGROUND OF INVENTION

Inhibitors of the Hedgehog (Hh) molecular signaling pathway (HhP) haveemerged in recent years as a promising new class of potentialtherapeutics for cancer treatment. Numerous drug discovery efforts haveresulted in the identification of a wide variety of small molecules thattarget different members of this pathway, including Smoothened (Smo),Sonic hedgehog protein (Shh), and Glioma-Associated Oncogene Homolog I,II, and III (Gli1, Gli2, and Gli3). Smo inhibitors have now enteredhuman clinical trials, and successful proof-of-concept studies have beencarried out in patients with defined genetic mutations in the Hhpathway. In fact, the first Smo inhibitor was approved by the FDA inearly 2012 for use in treatment of patients with advanced basal cellcarcinoma (vismodegib, marketed as ERIVEDGE™ from Roche/Genentech),validating the commercial validity of using drugs to modulate thispathway.

Activation of the (HhP) has been implicated in the development ofcancers in various organs, including brain, lung, mammary gland,prostate, and skin. Basal cell carcinoma, the most common form ofcancerous malignancy, has the closest association with hedgehogsignaling. Loss-of-function mutations in Patched and activatingmutations in Smo have been identified in patients with this disease(Sahebjam et al., “The Utility of Hedgehog Signaling Pathway Inhibitionfor Cancer,” The Oncologist, 2012; 17:1090-1099).

As an antifungal, the mechanism of action of itraconazole is the same asthe other azole antifungals, inhibiting the fungal-mediated synthesis ofergosterol. However, itraconazole has been discovered to haveanti-cancer properties. Itraconazole inhibits angiogenesis and Hhsignaling and delays tumor growth in murine prostate cancer xenograftmodels. Itraconazole appears to act on the essential Hh pathwaycomponent Smo in a mode that is different than the drug vismodegib, bypreventing the ciliary accumulation of Smo normally caused by Hhstimulation and has a much shorter half-life, which may be the reason ithas less side effects than vismodegib.

Prostate cancer rates are higher and prognoses are poorer in developedcountries than in the rest of the world. Prostate cancer is theninth-most-common cancer in the world, but is the number-one non-skincancer in men from the United States. Prostate cancer affects a largepercent of American men, sometimes resulting in death. In patients whoundergo treatment of prostate cancer, the most important clinicalprognostic indicators of disease outcome are stage, pre-therapyprostate-specific antigen (PSA) level, and Gleason score. In general,the higher the grade and the stage of prostate cancer, the poorer theprognosis. Nomograms can also be used to calculate the estimated risk ofthe individual patient. Some but not all prostate cancers appear to havean up-regulation of the Hh molecular pathway (U.S. Patent ApplicationPublication No. 20120083419, Altaba et al. “Method and Compositions forInhibiting Tumorigenesis”, which is incorporated herein by reference inits entirety).

It would be advantageous to have available a prognostic tool orbiomarker with proven ability to identify and distinguish, as early aspossible, those cancer patients who are likely to respond to HhPinhibitor treatment from those patients that are not, so that HhPinhibitor treatments can be provided to those patients for which an HhPinhibitor will be effective and alternative treatment modalities can beprovided to those for which an HhP inhibitor will be ineffective or lesseffective than other available treatments.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns methods for treating proliferationdisorders, such as prostate cancer, basal cell carcinoma, lung cancer,and other cancers, with a Hedgehog pathway (HhP) inhibitor, and methodsfor monitoring subjects undergoing such treatments based on biomarkersand other criteria predictive of efficacy.

Some aspects of the invention concern methods for prognosticating anoutcome of prostate cancer treatment with HhP inhibitor therapy, and fordetermining the efficacy of HhP inhibitor therapy, based on post-therapyprostate-specific antigen. Unlike the majority of prostate cancer drugs,which target androgens in order to lower testosterone levels,itraconazole's effect is androgen-independent.

A non-comparative, randomized, phase II study was conducted evaluatingthe antitumor efficacy of two doses of oral itraconazole in men withmetastatic prostate cancer. Based on the analysis described in FIGS.1-3, an increase in PSA post-treatment was identified as a marker forresponders to itraconazole therapy. The inventors identified patientswho exhibited a PSA increase of <25% at 4 weeks post-treatment withitraconazole as those patients who were the best responders to high-doseitraconazole therapy as far as PSA progression free survival (PPFS) andProgression Free Survival (PFS). Patients who were able to achieveplasma levels of HhP inhibitor (e.g., itraconazole) of >1000 ng/ml at 4weeks, along with the above-mentioned <25% PSA increase, are a targetsubpopulation of patients who can be pre-selected for treatment with HhPinhibitors as an enrichment strategy for clinical testing with theseagents in prostate cancer patients (FIG. 1). Retrospective analysisshowed that 14 of 15 high risk patients (PSA doubling times of less than6 months) on high-dose itraconazole demonstrated PSA increases of <25%at 4 weeks which translated into significant improvements in ProgressionFree Survival (FIG. 2). K-M Analysis based on PSA change at 4 weeks isshown in FIG. 3. Surprisingly, this effect was observed to continuebeyond the 4 week time point.

PSA levels are known to be a function of androgen activity. As androgenactivity increases (such as results from supplemental testosteronetherapy), the higher the PSA. As androgen activity decreases (such asresults from androgen deprivation therapy, anti-androgens, etc.), thelower the PSA. Because itraconazole has no effect on androgen synthesis,plasma levels or receptor activity, it was not expected thatitraconazole would have any significant effect on PSA. Additionally,since itraconazole is an inhibitor of the HhP and is not significantlycytotoxic (unlike chemotherapy), there was no reason to expect anysignificant effect on PSA because itraconazole does not kill the cancercells (and, hence, eliminating their contribution to PSA levels).Dendreon's PROVENGE® cancer vaccine is an example of anti-prostatecancer agent that has no effect on PSA or PFS but provides improvedoverall survival. In contrast, in the case of itraconazole, theinventors discovered unexpectedly that a reduction in PSA rise of </=25%is associated with a significant PFS and PSA-PFS. Furthermore, since HhPupregulation cannot be easily measured in men who have undergone radicalprostatectomy with no radiographic evidence of recurrence or metastaticdisease but who have rising PSA, the ability to use the PSA rise of<1=25% provides a way to determine sensitivity to a HhP inhibitor in asituation where the clinicians cannot be sure that the PSA is associatedwith upregulation of the HhP. Thus, unless the HhP is upregulated, itappears that an HhP inhibitor is not therapeutic, meaning that normalactivity of the HhP in a PSA cell is not going to render it susceptibleto HhP inhibitor treatment.

Surprisingly, the inventors found that the plasma concentrations ofitraconazole required to show a clinical benefit in humans with cancerare significantly greater than the typical levels for antifungalefficacy. Shi W. et al. reported that antifungal potency is determinedby structure unrelated to the inhibition of the HhP (Shi W. et al.,“Itraconazole Side Chain Analogues: Structure-Activity RelationshipStudies for Inhibition of Endothelial Cell Proliferation, VascularEndothelial Cell Growth Factor Receptor 2 (VEGFR2) Glycosylation, andHedgehog Signaling,” J. Med. Chem., 2011, 54:7363-7374, which isincorporated herein by reference in its entirety); thus, the use ofhigher doses of HhP inhibitors such as itraconazole to treat systemicfungal infections in cancer patients did not support or suggest that thedose required to treat cancer was in the range of high dose antifungaltherapy.

Furthermore, the determination of the minimum trough level to achieve aneffect on proliferation disorders such as cancer was not predicted fromthe in vitro studies by Shi W. et al because of these considerations:(i) itraconazole has multiple anti-cancer properties, includinganti-angiogenic, mTOR (mammalian target of rapamycin) inhibition, andanti-hedgehog; therefore, the in vitro studies of the HhP oranti-angiogenic effects, for example, were not sufficient to predictdosing or plasma levels; (ii) after a number of days of dosing,sufficient to achieve a steady state, the tissue concentration ofitraconazole is known to be a multiple of plasma levels; and (iii)unlike the antifungal effects, the major metabolite of itraconazole(hydroxy-itraconazole) is not equipotent as a HhP inhibitor but it doesseem to have significant effects making extrapolation to in vivo plasmalevels that are effective in cancer impossible to predict.

One aspect of the invention concerns a method for treating aproliferation disorder in a subject, such as prostate cancer, basal cellcarcinoma, lung cancer, and other cancers, comprising orallyadministering a composition comprising a Hedgehog pathway (HhP)inhibitor to the subject, wherein the composition is orally administeredin an effective amount to achieve a plasma trough level of at leastabout 1,000 ng/mL of the HhP inhibitor. Optionally, the method oftreatment includes monitoring the proliferation disorder in the subjectto determine whether there has been a clinical response to HhP inhibitortreatment.

Another aspect of the invention concerns a method of prognosticating anoutcome of prostate cancer treatment with a Hedgehog pathway (HhP)inhibitor therapy in a subject, comprising comparing the level ofprostate-specific antigen (PSA) in a sample obtained from the subjectfollowing HhP inhibitor therapy with a reference level of PSA, whereinthe level of PSA in the sample compared to the reference level of PSA isprognostic for an outcome of treatment with the HhP inhibitor.

Another aspect of the invention concerns a method of determining theefficacy of Hedgehog pathway (HhP) inhibitor therapy for prostate cancerin a human subject, comprising measuring prostate-specific antigen (PSA)level in a sample obtained from the subject following initiation of HhPinhibitor therapy, wherein a measured PSA level compared to a firstreference PSA level at initiation of HhP inhibitor therapy is indicativeof efficacy, and wherein a measured PSA level compared to a secondreference PSA level is indicative of a lack of efficacy.

Another aspect of the invention concerns a method for treating prostatecancer in a subject, comprising administering Hedgehog pathway (HhP)inhibitor therapy to the subject; and carrying out a method of theinvention (i.e., a method of prognosticating an outcome of prostatecancer treatment with a HhP inhibitor therapy, or a method ofdetermining the efficacy of HhP inhibitor therapy).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: PSA increase at four weeks as a screening tool for responders toitraconazole therapy. The data reflect analysis of parameters that areindicative of successful administration of itraconazole therapy toprostate cancer patients based on PSA response. The inventors identifiedpatients who exhibited a PSA increase of <25% at 4 weeks post-treatmentwith itraconazole as those patients who were the best responders tohigh-dose itraconazole therapy as far as PSA progression free survival(PPFS) and Progression Free Survival (PFS). Patients who were able toachieve plasma levels of itraconazole of >1000 ng/ml at 4 weeks, alongwith the above-mentioned <25% PSA increase, are a target subpopulationof patients who can be pre-selected for treatment with itraconazole asan enrichment strategy for clinical testing of itraconazole in prostatecancer patients.

FIG. 2: In this retrospective analysis, 14 of 15 high risk patients (PSAdoubling times of less than 6 months) on high-dose itraconazoledemonstrated PSA increases of <25% at 4 weeks which translated intosignificant improvements in Progression Free Survival.

FIG. 3: K-M Analysis based on PSA change at 4 weeks. PFS (radiographic)in high-dose group.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention concerns a method for treating aproliferation disorder in a subject, comprising orally administering acomposition comprising a Hedgehog pathway (HhP) inhibitor to thesubject, wherein the composition is orally administered in an effectiveamount to achieve a plasma trough level of at least about 1,000 ng/mL ofthe HhP inhibitor.

In some embodiments, the composition is administered in an effectiveamount to achieve a plasma trough level of at least 1,000 ng/mL of theHhP inhibitor. In some embodiments, the composition is administered inan effective amount to achieve a plasma trough level of at least about1,000 ng/mL of the HhP inhibitor after about 4 weeks of initiation oftreatment with the HhP inhibitor. In some embodiments, the compositionis administered in an effective amount to achieve a plasma trough levelof at least about 1,000 ng/mL of the HhP inhibitor within about 2 weeksafter initiation of treatment, and to maintain the plasma trough levelof at least about 1,000 ng/mL of the HhP inhibitor for the duration ofthe treatment.

Any inhibitor of the HhP may be used. In some embodiments, the HhPinhibitor targets the Smoothened (Smo) protein of the HhP pathway,acting on Smo, for example, by binding to it. In some embodiments, theHhP inhibitor is cyclopamine-competitive. In some embodiments, the HhPinhibitor comprises itraconazole, or a pharmaceutically acceptable salt,prodrug, or active metabolite thereof. In some embodiments, the HhPinhibitor is a purified stereoisomer of itraconazole (non-racemicmixture), or an itraconazole analogue in which the sec-butyl side chainhas been replaced with one or more moieties, relative to itraconazole.In some embodiments, the HhP inhibitor is cyclopamine-competitive. Insome embodiments, the HhP inhibitor is non-cyclopamine-competitive. Insome embodiments, the HhP inhibitor is cyclopamine-competitive and theproliferation disorder is prostate cancer, basal cell carcinoma, or lungcancer.

The HhP inhibitor may be formulated for the desired delivery route.Furthermore, achieving the desired level of HhP inhibitor can beenhanced by the use of formulations with greater bioavailability. Forexample, the HhP inhibitor may be administered in a composition such asSUBA® formulation of itraconazole, or a pharmaceutically acceptablesalt, prodrug, or active metabolite thereof. In some embodiments, an HhPinhibitor such as itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, is administered ina SUBA® formulation at a dose in the range of 100 mg to 600 mg per day.In some embodiments, 150 mg of an HhP inhibitor such as itraconazole, ora pharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, is administered in a SUBA® formulation two or moretimes per day. In some embodiments, 200 mg of an HhP inhibitor such asitraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite thereof, is administered in a SUBA®formulation two or more times per day.

In some embodiments, the HhP inhibitor therapy comprises oraladministration of a capsule, tablet, or suspended powder (liquidsuspension), or liquid solution of 50 mg of the itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, twice per day. In some embodiments, the SUBA®formulation is a SUBA-CAP™ formulation.

Optionally, the treatment method further comprises measuring the plasmalevel of the HhP inhibitor, or a metabolite thereof, in the subject oneor more times. In some embodiments, the measuring is carried out one ormore times about 4 weeks after initiation of treatment with the HhPinhibitor.

In some embodiments, the method includes measuring the plasma level ofthe HhP inhibitor, or a metabolite thereof, one or more times in aperiod of time from about 4 weeks to about 12 weeks. Optionally, themethod further comprises increasing a subsequent dose of the HhPinhibitor if the plasma trough level of at least about 1,000 ng/mL ofthe HhP inhibitor is not maintained. Optionally, the method may furthercomprise reducing a subsequent dose of an HhP inhibitor if the plasmatrough level at about 4 weeks is at least 1000 ng/mL and the subject isexperiencing one or more side effects.

Various dosing regimens may be utilized. In some embodiments, the HhPinhibitor is administered at least once daily. In some embodiments, theHhP inhibitor is administered at least twice daily. In some embodiments,the duration of treatment with the HhP inhibitor is in the range ofabout 4 weeks to about 24 weeks. In some embodiments, once achieved, aplasma trough level of at least about 1,000 ng/mL of HhP inhibitor ismaintained throughout the therapy.

In some embodiments, the proliferation disorder is a cancer, such asprostate cancer, basal cell carcinoma, lung cancer, or other cancer.

In some embodiments, the proliferation disorder is prostate cancer andthe method further comprises comparing the level of prostate-specificantigen (PSA) in a sample obtained from the subject followingadministration of the HhP inhibitor with a reference level of PSA,wherein the level of PSA in the sample compared to the reference levelof PSA is prognostic for an outcome of treatment with the HhP inhibitor.In some embodiments, a PSA level increase of less than about 25%relative to the PSA level at initiation of HhP inhibitor treatment isindicative of efficacy and a PSA level increase of about 25% or greateris indicative of a lack of efficacy. In some embodiments, the subjecthas a PSA level increase of less than about 25% after about 4 weeks onHhP inhibitor treatment relative to the PSA level at initiation of HhPinhibitor treatment.

In some embodiments, the sample is obtained from the subject within 4 to12 weeks after initiation of HhP inhibitor therapy.

In some embodiments, the method further comprises obtaining the samplefrom the subject after said administering.

In the case of prostate cancer, in some embodiments, the method furthercomprises maintaining HhP inhibitor therapy if the measured level of PSAis indicative of efficacy.

In the case of prostate cancer, in some embodiments, the method furthercomprises ceasing treatment with the HhP inhibitor if the measured levelof PSA is indicative of a lack of efficacy. Optionally, the methodfurther comprises administering a treatment for the prostate cancerother than an HhP inhibitor. In some embodiments, the treatmentcomprises one or more from among radiation therapy, hormone therapy,chemotherapy, immunotherapy, surgery, cryosurgery, high-intensityfocused ultrasound, and proton beam radiation therapy.

In the case of prostate cancer, in some embodiments, the method furthercomprises increasing the dose of the HhP inhibitor and/or frequency ofdose of the HhP inhibitor if the measured level of PSA is indicative ofa lack of efficacy.

In the case of prostate cancer, in some embodiments, the method furthercomprises decreasing the dose of the HhP inhibitor and/or frequency ofdose of the HhP inhibitor if the measured level of PSA is indicative ofefficacy but the subject is experiencing one or more adverse effects.

In the case of prostate cancer, in some embodiments, the PSA levelmeasured is the level of total PSA (free (unbound) PSA and bound PSA).In some embodiments, the PSA level measured is PSA doubling time.

In the case of prostate cancer, in some embodiments, the PSA proteinlevel is measured, using methods such as radioimmunoassay (MA),immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay(ELISA), dot blot, slot blot, enzyme-linked immunosorbent spot (ELISPOT)assay, Western blot, peptide microarray, surface plasmon resonance,fluorescence resonance energy transfer, bioluminescence resonance energytransfer, fluorescence quenching fluorescence, fluorescencepolarization, mass spectrometry (MS), high-performance liquidchromatography (HPLC), high-performance liquid chromatography/massspectrometry (HPLC/MS), high-performance liquid chromatography/massspectrometry/mass spectrometry (HPLC/MS/MS), capillary electrophoresis,rod-gel electrophoresis, or slab-gel electrophoresis.

In some embodiments, the PSA DNA or mRNA level is measured using methodssuch as Northern blot, Southern blot, nucleic acid microarray,polymerase chain reaction (PCR), real time-PCR (RT-PCR), nucleic acidsequence based amplification assay (NASBA), or transcription mediatedamplification (TMA).

In the case of prostate cancer, in some embodiments, the PSA activitylevel is measured.

Optionally, in the case of prostate cancer, the treatment method furthercomprises monitoring the PSA level in the subject, comprising comparingthe PSA level in multiple samples with the reference level of PSA,wherein the samples are obtained from the subject over time, followingHhP inhibitor treatment.

In some embodiments, the method of treatment further comprises obtainingthe sample from the subject. In some embodiments, the sample is a serumsample.

The method of treatment may include monitoring the proliferationdisorder in the subject to determine whether there has been a clinicalresponse to HhP inhibitor treatment. In some embodiments, the methodfurther comprises monitoring the proliferation disorder in the subject,wherein a lack of clinical response in the proliferation disorder to thetreatment is indicative that the plasma trough level of the HhPinhibitor should be increased further above about 1000 ng/mL, andwherein the occurrence of a clinical response and a plasma trough levelof the HhP inhibitor substantially higher than about 1000 ng/mLindicates that one or more subsequent doses of the HhP inhibitor can bereduced. In some embodiments, the method further comprises monitoringthe proliferation disorder in the subject, wherein a lack of clinicalresponse in the proliferation disorder to the treatment, after aboutfour weeks of said administering, is indicative of a need to increasethe dose, and/or frequency of the dose, of the HhP inhibitor.Optionally, the method further comprises subsequently administering theHhP inhibitor to the subject at the increased dose and/or frequency. Insome embodiments, the method further comprises monitoring theproliferation disorder in the subject, wherein the occurrence of aclinical response in the proliferation disorder to the treatment, afterabout four weeks of said administering, is indicative of a need todecrease the dose, and/or frequency of the dose, of the HhP inhibitor.Optionally, the method further comprises subsequently administering theHhP inhibitor to the subject at a decreased dose and/or frequency.

In some embodiments, the monitoring comprises visual inspection,palpation, imaging, assaying the presence, level, or activity of one ormore biomarkers associated with the proliferation disorder in a sampleobtained from the subject, or a combination of two or more of theforegoing. In some embodiments, the monitoring comprises monitoring atleast one of the following parameters: tumor size, rate of change intumor size, hedgehog levels or signaling, appearance of new tumors, rateof appearance of new tumors, change in symptom of the proliferationdisorder, appearance of new symptom associated with the proliferationdisorder, quality of life (e.g., amount of pain associated with theproliferation disorder), or a combination of two or more of theforegoing.

As indicated above, the inventors found that the plasma concentrationsof itraconazole required to show a clinical benefit in humans withcancer are significantly greater than the typical levels for antifungalactivity. In particular, the minimum plasma trough level after 4 weeksof therapy required to have a clinically significant effect was at least1000 ng/ml. Achieving these levels of itraconazole is enhanced by theuse of formulations with greater bioavailability such as SUBA-CAP™formulation. Nevertheless, there can be side-effects peculiar to suchhigh doses such as hypertension, peripheral edema, and hypokalemia,which seem to be a result of an increased production ofmineralocorticoid. These side effects associated with these high dosesof itraconazole can be effectively managed by giving a selectivemineralocorticoid antagonist, such as eplerenone. Accordingly, in someembodiments, the method further comprises administering eplerenone orother mineralocorticoid inhibitor. In some embodiments, the subject issuffering from an adverse effect selected from hypertension, peripheraledema, and hypokalemia, and wherein the mineralocorticoid inhibitor isadministered in an amount effective to treat the adverse effect.

In some embodiments, the subject has a fungal infection. In otherembodiments, the subject does not have a fungal infection.

In some embodiments, the subject has a fungal infection selected fromBlastomycosis, Histoplasmosis, Candidiasis, and Aspergillosis. In otherembodiments, the subject does not have a fungal infection selected fromamong Blastomycosis, Histoplasmosis, Candidiasis, and Aspergillosis.

In some embodiments, the subject has received no prior chemotherapy totreat the proliferation disorder.

In some embodiments, the subject is administered no steroid during theduration of the treatment.

In some embodiments, the subject is administered no agent that interactswith CYP3A4 during the duration of the treatment.

The present invention also concerns methods for prognosticating anoutcome of prostate cancer treatment with a Hedgehog pathway (HhP)inhibitor therapy, and for determining the efficacy of HhP inhibitortherapy, based on post-therapy prostate-specific antigen.

One aspect of the invention concerns a method of prognosticating anoutcome of prostate cancer treatment with a Hedgehog pathway (HhP)inhibitor therapy in a subject, comprising comparing the level ofprostate-specific antigen (PSA) in a sample obtained from the subjectfollowing HhP inhibitor therapy with a reference level (predeterminedlevel) of PSA, wherein the level of PSA in the sample compared to thereference level of PSA is prognostic for an outcome of treatment withthe HhP inhibitor. In some embodiments, the reference level is the PSAlevel in the subject at initiation of HhP inhibitor therapy. In someembodiments, the method comprises monitoring the PSA level in thesubject, comprising comparing the PSA level in multiple samples with thereference level of PSA, wherein the samples are obtained from thesubject over time, following HhP inhibitor therapy.

Another aspect of the invention concerns a method of determining theefficacy of Hedgehog pathway (HhP) inhibitor therapy for prostate cancerin a human subject, comprising measuring prostate-specific antigen (PSA)level in a sample obtained from the subject following initiation of HhPinhibitor therapy, wherein a measured PSA level compared to a firstreference PSA level (first predetermined level) at initiation of HhPinhibitor therapy is indicative of efficacy, and wherein a measured PSAlevel compared to a second reference PSA level (second predeterminedlevel) is indicative of a lack of efficacy. In some embodiments, themethod comprises monitoring the PSA level in the subject, comprisingmeasuring the PSA level in multiple samples obtained from the subjectover time, following HhP inhibitor therapy (e.g., at one or more of 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,12 weeks or longer following initiation of HhP therapy). In someembodiments, a sample is obtained at about 3 to 5 weeks and/or at about11 to 13 weeks following initiation of HhP inhibitor therapy. In someembodiments, a sample is obtained at about 4 weeks and/or at about 12weeks following initiation of HhP inhibitor therapy.

In some embodiments of the methods of the invention, a PSA levelincrease of less than about 25% relative to the PSA level at initiationof HhP inhibitor therapy is indicative of efficacy and a PSA levelincrease of about 25% or greater is indicative of a lack of efficacy.

In some embodiments of the methods of the invention, the HhP inhibitorcomprises itraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite thereof. For example, the HhPinhibitor may comprise or consist of a SUBA® formulation (Mayne PharmaInternational Pty Ltd., e.g., the SUBACAP™ formulation) of itraconazole(see, for example, U.S. Patent Application Publication No. 20030225104(Hayes et al., “Pharmaceutical Compositions for Poorly Soluble Drugs,”issued as U.S. Pat. No. 6,881,745), which is a solid dispersion whereinitraconazole is associated with acidic molecules and the formulationallows for improved absorption. In some embodiments, the HhP inhibitor,such as a SUBA® formulation, is administered to the subject at a dose inthe range of 100 mg to 600 mg per day.

In some embodiments, the HhP inhibitor is administered intravenously orlocally (e.g., by direct injection) to a prostate cancer lesion ortumor. In some embodiments, the HhP inhibitor is administered orally,e.g., in capsule, tablet, suspended powder (liquid suspension), orliquid solution form. In some embodiments, the HhP inhibitor is orallyadministered (e.g., in capsule, tablet, suspended powder (liquidsuspension), or liquid solution form) in an amount comprising orconsisting of about 25 mg to about 100 mg per dose twice a day. In someembodiments, the HhP inhibitor is orally administered (e.g., in capsule,tablet, suspended powder (liquid suspension), or liquid solution form)in an amount comprising or consisting of 50 mg per dose twice a day.

In some embodiments of the methods of the invention, the sample isobtained from the subject within 4 to 6 weeks after initiation of HhPinhibitor therapy.

In some embodiments of the methods of the invention, the method furthercomprises administering the HhP inhibitor to the subject, and obtainingthe sample from the subject after said administering.

In some embodiments of the methods of the invention, the method furthercomprises maintaining HhP inhibitor therapy if the measured level of PSAis indicative of efficacy.

In some embodiments of the methods of the invention, the method furthercomprises withholding HhP inhibitor therapy if the measured level of PSAis indicative of a lack of efficacy. Withholding HhP inhibitor therapymay include watchful waiting or active surveillance. Optionally, themethod further comprises administering one or more treatments for theprostate cancer other than an HhP inhibitor. Examples of prostate cancertreatments include, but are not limited to, radiation therapy, hormonetherapy, chemotherapy, immunotherapy, surgery (surgical excision/removalof cancerous tissue, e.g., open or laparoscopic prostatectomy),cryosurgery, high-intensity focused ultrasound, and proton beamradiation therapy.

It should be understood that indications of HhP inhibitor therapyefficacy or lack of efficacy can be specific to the dose and/orfrequency of the dose administered. In this way, the invention providesa method for determining a dose of HhP inhibitor suitable foradministration to a subject for treatment of prostate cancer. Thisinvolves carrying out a method of prognosticating an outcome of prostatecancer treatment or determining efficacy of an HhP inhibitor therapy asdescribed herein, and determining an effective dose of HhP inhibitorbased on the comparison of PSA level measured in a sample obtainedfollowing a dosage level and/or dose frequency change to a reference PSAlevel.

For example, it is possible to administer a dose of HhP inhibitor at onelevel and/or one frequency and not observe a PSA response, butadminister a dose at a different (greater) level and/or frequency andobserve a PSA response. Therefore, the dose level and/or frequency ofdosing may affect whether an HhP inhibitor works or does not work.Consequently, if a lack of efficacy is indicated based on PSA level atone dose and/or one frequency of the HhP inhibitor, before withholdingthe HhP therapy and/or administering an alternative (non-HhP inhibitor)treatment for the prostate cancer, it may be desirable to modulate(e.g., increase) the dosage and/or frequency of the HhP inhibitor and,optionally, obtain one or more subsequent samples and measure the PSAlevel in the sample(s) and compare the measured level to the referencelevel to make another determination of prognosis orefficacy/non-efficacy at the different dosage and/or frequency.Accordingly, in some embodiments of the methods of the invention, themethod further comprises increasing the dose of the HhP inhibitor and/orfrequency of dose of the HhP inhibitor if the measured level of PSA isindicative of a lack of efficacy. This may be repeated one or more timesuntil efficacy of that dosage regimen is indicated based on measuredlevel of PSA relative to the reference level (e.g., as a dose titrationusing reference PSA level as a guide). Optionally, at any point in theprocess, the HhP inhibitor can be withheld and, optionally, analternative (non-HhP inhibitor) treatment administered to the subject.

Alternatively, if a subject does achieve a PSA level indicative ofefficacy at one dose level and/or frequency, but the subject experiencesone or more side effects, then the dose level and/or frequency of dosemay be subsequently decreased. One or more samples may then be obtained,PSA level measured, and compared to a reference level to ensure that themeasured PSA level at the decreased dose and/or frequency remainsindicative of efficacy. Again, the PSA level may be used as a biomarkeror guide for optimal dosing of subsequent administrations with the HhPinhibitor. Accordingly, in some embodiments of the methods of theinvention, the method further comprises decreasing the dose of the HhPinhibitor and/or frequency of dose of the HhP inhibitor if the measuredlevel of PSA is indicative of efficacy but the subject is experiencingone or more side effects. This may be repeated one or more times untilthe side effects are reduced or eliminated without compromising efficacyof that dosage regimen based on PSA level. Optionally, at any point inthe process, the HhP inhibitor can be withheld and, optionally, analternative (non-HhP inhibitor) treatment administered to the subject.This may be desirable if the side effects are not manageable withoutcompromising efficacy.

As indicated above, an aspect of the invention is a method fordetermining a dose of HhP inhibitor suitable for administration to asubject for treatment of prostate cancer, comprising measuring a PSAlevel in a sample obtained from the subject following HhP inhibitoradministration (e.g., at about 4 weeks and/or about 12 weeks afterinitiation of HhP inhibitor therapy); and determining an effective doseof the HhP inhibitor based on comparison of the measured PSA level to areference level of PSA (e.g., a PSA level increase of less than about25% relative to the PSA level at initiation of HhP inhibitor therapy).By way of example, 50 mg of an HhP inhibitor may be administeredincrementally to a subject to establish efficacy by increasing the dose(adjusting the amount and/or frequency of subsequent doses upward) ifthe subject does not respond or decreasing the dose (adjusting theamount and/or frequency downward) if it is too toxic. In the case of aSUBA® formulation of an azole antifungal drug, for example, such as aSUBACAP™ formulation, a dose may be titrated up or down such that thedose is within the range of 100 mg to 600 mg of SUBA™ formulation perday usually in divided doses administered twice daily. The high end ofthe range may be used for example to obtain rapid trough levels onday-one or day-two and then the dose may be reduced (in amount and/orfrequency), or for some prostate cancers, it may be determined that amore potent dose is required.

In some embodiments of the methods of the invention, the PSA level isthe level of total PSA (free (unbound) PSA and bound PSA). In someembodiments of the methods of the invention, the PSA level is PSAdoubling time.

In the methods of the invention, the determined PSA level may representthe amount of PSA protein, the amount of nucleic acid (DNA or mRNA)encoding PSA, or the amount of PSA activity. In some embodiments, thePSA protein level is measured by radioimmunoassay (MA),immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay(ELISA), dot blot, slot blot, enzyme-linked immunosorbent spot (ELISPOT)assay, Western blot, peptide microarray, surface plasmon resonance,fluorescence resonance energy transfer, bioluminescence resonance energytransfer, fluorescence quenching fluorescence, fluorescencepolarization, mass spectrometry (MS), high-performance liquidchromatography (HPLC), high-performance liquid chromatography/massspectrometry (HPLC/MS), high-performance liquid chromatography/massspectrometry/mass spectrometry (HPLC/MS/MS), capillary electrophoresis,rod-gel electrophoresis, or slab-gel electrophoresis. In someembodiments, the PSA mRNA level is measured by Northern blot, Southernblot, nucleic acid microarray, polymerase chain reaction (PCR), realtime-PCR (RT-PCR), nucleic acid sequence based amplification assay(NASBA), or transcription mediated amplification (TMA).

The sample obtained from the subject may be potentially any sampleharboring PSA protein or nucleic acids. The sample may be processedbefore or after the PSA biomarker is measured. In some embodiments ofthe methods of the invention, the sample is a serum sample.

The methods of the invention may further comprise obtaining the samplefrom the subject, such as by withdrawing blood or by tissue biopsy.

The methods of the invention may further comprise identifying thesubject as having prostate cancer (e.g., based on one or morebiomarkers, signs, symptoms, biopsy, etc.) before initiating HhPtherapy.

In some embodiments, prior to initiation of treatment with the HhPinhibitor, the subject has undergone treatment for the prostate cancerwith a non-HhP inhibitor. For example, the HhP inhibitor may beadministered as a second line, third line, or fourth line therapy.

There are other tools available to help predict outcomes in prostatecancer treatment, such as pathologic stage and recurrence after surgeryor radiation therapy. Most combine stage, grade, and PSA level, and somealso add the number or percent of biopsy cores positive, age, and/orother information. The methods of the invention may be used in additionto, or as an alternative to, methods for prognosticating prostatecancer, such as D'Amico classification, the Partin tables, the Kattannomograms, and the UCSF Cancer of the Prostate Risk Assessment (CAPRA)score.

Another aspect of the invention concerns a method for treating prostatecancer in a subject, comprising administering Hedgehog pathway (HhP)inhibitor therapy to the subject; and carrying out a method of theinvention (i.e., a method of prognosticating an outcome of prostatecancer treatment with a HhP inhibitor therapy, or a method ofdetermining the efficacy of HhP inhibitor therapy).

Patient Selection

Optionally, subjects in need of treatment (or further treatment) of aproliferation disorder such as prostate cancer, basal cell carcinoma,lung cancer, or other cancer, may be selected as an individualparticularly suitable for treatment with an HhP inhibitor, based on Hhlevels or signaling, which may be assessed directly or indirectly bymeasuring a biomarker (an HhP biomarker) that represents the HhP signalitself or a modulator of the HhP signal (inducer or inhibitor). If thebiomarker is an inhibitor of the HhP signal, and the level of theinhibitor is below normal, an assumption may be made that the HhP signalis elevated above normal. Likewise, if the biomarker is an inhibitor ofthe HhP signal, and the level of the inhibitor is above normal, anassumption may be made that the HhP signal is reduced below normal. Ifthe biomarker is an inducer of the HhP signal, and the level of theinducer is below normal, an assumption may be made that the HhP signalis reduced below normal. Likewise, if the biomarker is an inducer of theHhP signal, and the level of the biomarker is above normal, anassumption may be made that the HhP signal is elevated above normal.Optionally, the accuracy of the aforementioned assumptions may beconfirmed by measuring HhP signaling directly or by measuring otheradditional HhP biomarkers.

Hh levels or signaling may be assessed by measuring an HhP protein, or anucleic acid encoding an HhP protein such as an HhP ligand thatactivates the pathway and/or an upstream or downstream component(s) ofthe HhP, e.g., a receptor, activator or inhibitor of hedgehog. Ligandsof the mammalian HhP include Sonic hedgehog (SHH), desert hedgehog(DHH), and Indian hedgehog (DHH). Activation of the HhP leads to nucleartranslocation of glioma-associated oncogene homolog (Gli) transcriptionfactors, and the levels of these transcription factors may be assessedas well (e.g., Gli1, Gli2, Gli3, or a combination or two or more of theforegoing).

Any of the aforementioned biomarkers can be detected in a sampleobtained from the subject such as blood, urine, circulating tumor cells,a tumor biopsy, or a bone marrow biopsy. These biomarkers can also bedetected by systemic administration of a labeled form of an antibody toa biomarker followed by imaging with an appropriate imaging modality.The measured level in the sample may be compared to a reference levelsuch as a normal level representative of constitutive expression of thebiomarker or a normal level of HhP signaling, or a level that waspreviously measured in a sample obtained from the subject (e.g., in asample obtained from the subject at an earlier time in the treatmentregimen or before the subject developed the proliferation disorder). Ifthe HhP biomarker is upregulated (elevated) relative to the referencelevel, then the subject can be selected for treatment with an HhPinhibitor such as itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, and administrationof the HhP inhibitor to the subject may proceed. Furthermore, asdescribed below, the proliferation disorder may then be monitored for aclinical response by obtaining another sample from the subject,measuring the biomarker, and comparing the measured level to the levelmeasured in the sample that was obtained previously. Multiple samplesmay be obtained and measurements determined and compared during thecourse of the treatment to monitor the proliferation disorder andclinical response to the treatment over time.

Monitoring a Proliferation Disorder

Because not every proliferation disorder may be immediately responsiveto every dosage regimen with an HhP inhibitor, even in the therapeuticrange of at least about 1000 ng/mL, it may be desirable to monitor theproliferation disorder in the subject for the presence or absence of aresponse to the HhP inhibitor treatment. The plasma trough level of atleast about 1000 ng/ml ensures an empirical trial of HhP inhibitor ismore likely to be effective but it may take higher levels to beeffective and in some subjects no matter what the dose, the HhPinhibitor is not effective, perhaps because the HhP is not up-regulatedor there are mutations that make the HhP inhibitor ineffective inblocking the up-regulation.

Accordingly, in some embodiments, the method further comprisesmonitoring the proliferation disorder for the presence or absence of aresponse to the HhP inhibitor treatment. In some embodiments, the methodfurther comprises monitoring the proliferation disorder in the subject,wherein a lack of clinical response in the proliferation disorder to thetreatment is indicative that the plasma trough level of the HhPinhibitor should be increased further above about 1000 ng/mL, andwherein the occurrence of a clinical response and a plasma trough levelof the HhP inhibitor substantially higher than about 1000 ng/mLindicates that one or more subsequent doses of the HhP inhibitor can bereduced. In some embodiments, the method further comprises monitoringthe proliferation disorder in the subject, wherein a lack of clinicalresponse in the proliferation disorder to the treatment, after aboutfour weeks of said administering, is indicative of a need to increasethe dose, and/or frequency of the dose, of the HhP inhibitor. In someembodiments, the method further comprises monitoring the proliferationdisorder in the subject, wherein the occurrence of a clinical responsein the proliferation disorder to the treatment, after about four weeksof said administering, is indicative of a need to decrease the dose,and/or frequency of the dose, of the HhP inhibitor.

In some embodiments, the monitoring comprises visual inspection,palpation, imaging, assaying the presence, level, or activity of one ormore biomarkers associated with the proliferation disorder in a sampleobtained from the subject, or a combination of two or more of theforegoing, one or more times at various intervals of treatment toascertain whether the treatment is effectively treating theproliferation disorder in the subject (causing or contributing to aclinical response in the subject). For skin cancers such a basal cell ormalignant melanoma visual inspection can be with unaided eye. Visualinspection via colonoscopy may be utilized for colorectal cancers andprecancerous proliferation disorders such as polyps. Bronchoscopy may beused for lung cancer. Esophagoscopy may be used for esophageal cancersand precancers (e.g., Barret's esophagus). Gastroscopy may be used forgastric cancers. Cystoscopy may be used for bladder cancers andprecancerous proliferation disorders. Laparoscopy may be used forovarian cancers and endometriosis. Biomarkers such as PSA, PCA2 antigen,and Gli (Gli1, Gli2, Gli3, or a combination of two or three Gli) may beassayed. For example, a decreased level of expression of the Gli in thesample relative to a reference level (such as a baseline) is indicativeof a positive clinical response to the HhP inhibitor treatment(efficacy), and an increased level of expression of the Gli relative toa reference level (such as a baseline) is indicative of a negativeclinical response or lack of clinical response to the HhP inhibitortreatment (lack of efficacy). Examples of other tumor markers areprovided below.

Examples of imaging modalities that may be utilized include computedtomography (CT), magnetic resonance imaging (MM), ultrasound, x-ray, andnuclear medicine scans. Palpation may be conducted for lymph nodes,transrectal digital exam for prostatic cancers, and a pelvic exam forovarian cancers, abdominal palpation for liver cancers (primary ormetastatic).

In some embodiments, the monitoring comprises monitoring at least one ofthe following parameters: tumor size, rate of change in tumor size,hedgehog levels or signaling, appearance of new tumors, rate ofappearance of new tumors, change in symptom of the proliferationdisorder, appearance of a new symptom associated with the proliferationdisorder, quality of life (e.g., amount of pain associated with theproliferation disorder), or a combination of two or more of theforegoing.

As indicated above, the method for treating a proliferation disorder mayinclude monitoring the proliferation disorder in the subject followingadministration of the HhP inhibitor, wherein a lack of clinical responsein the proliferation disorder to the treatment is indicative that theplasma trough level of the HhP inhibitor should be increased furtherabove about 1,000 ng/mL, and wherein the occurrence of a clinicalresponse and a plasma trough level of the HhP inhibitor substantiallyhigher than about 1,000 ng/mL indicates that one or more subsequentdoses of the HhP inhibitor can be reduced.

In some embodiments, the treatment method further comprises monitoringthe proliferation disorder in the subject for a clinical response. Insome embodiments, the clinical response is tumor response and theResponse Evaluation Criteria In Solid Tumors (RECIST) may be used todefine when tumors in cancer patients improve (show a “clinicalresponse”), stay the same (“stabilize”), or worsen (“progress”) duringtreatment. In some embodiments, a decrease in tumor size is indicativeof improvement or clinical response, and an increase or no change in thesize of a tumor is indicative of a lack of clinical response. The siteof the tumor will depend upon the type of cancer. In basal cellcarcinoma, the tumor will be in the skin. The occurrence of a clinicalresponse to the treatment after a period of time (e.g., after about fourweeks of administering the HhP inhibitor) indicates that the HhPinhibitor dose, HhP inhibitor dose frequency, and choice of HhPinhibitor(s) currently being administered are satisfactory and thetreatment may proceed in the absence of any adverse effects of thetreatment. The HhP inhibitor dose and/or frequency of dose may bereduced if any adverse effects are observed. A lack of clinical responsein the proliferation disorder to the treatment, after about four weeksof administering the HhP inhibitor, can be indicative of a need tomodify the treatment regimen by increasing the dose of the HhPinhibitor, or increasing the frequency of the dosing of the HhPinhibitor, or administering an additional HhP inhibitor before, duringor after the HhP inhibitor currently being administered, or acombination of two or more of the foregoing. In some embodiments, one ormore additional HhP inhibitors are administered and the additional HhPinhibitor differs from the currently administered HhP inhibitor(s) inits mechanism of action by which it inhibits the HhP (e.g.,itraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite of itraconazole, and vismodegib, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite of vismodegib). Multiple samples may be obtained andmeasurements determined and compared during the course of the treatmentto monitor the proliferation disorder over time.

Monitoring may comprise visual inspection, palpation, imaging, assayingthe presence, level, or activity of one or more biomarkers associatedwith the proliferation disorder and/or clinical response in a sampleobtained from the subject, or a combination of two or more of theforegoing. Examples of biomarkers include Gli1, Gli2, Gli3, PSA, and theplasma level of HhP inhibitor or its metabolite.

In some embodiments, monitoring comprises monitoring at least one of thefollowing parameters: tumor size, rate of change in tumor size, hedgehoglevels or signaling, appearance of a new tumor, rate of appearance ofnew tumors, change in a symptom of the proliferation disorder,appearance of a new symptom associated with the proliferation disorder,quality of life (e.g., amount of pain associated with the proliferationdisorder), or a combination of two or more of the foregoing. Followingtreatment, a decrease in tumor size, decreased rate of tumor growth, ordecrease in hedgehog levels or signaling, or lack of appearance of newtumors, or decrease in rate of new tumors, or improvement of a symptomof the proliferation disorder, or lack of appearance of a new symptom ofthe proliferation disorder, or improvement in the quality of life canindicate a clinical response, i.e., that the selected HhP inhibitor(s)and treatment dosing regimen are satisfactory and do not need to bechanged (though the dose and/or frequency of administration could bereduced if an adverse reaction exists). Likewise, following treatment,an increase in tumor size, or increased rate of tumor growth or nochange in tumor size, or increase in hedgehog levels or signaling, orappearance of new tumors, or increase in rate of new tumors, orworsening of a symptom of the proliferation disorder, or appearance of anew symptom of the proliferation disorder, or a decrease in quality oflife can indicate a lack of clinical response to the treatment and canindicate a need to modify the treatment regimen by increasing the doseof the HhP inhibitor (assuming that any adverse reaction, if present, ismanageable), or increasing the frequency of the dosing of the HhPinhibitor (again, assuming that any adverse reaction, if present, ismanageable), or administering an additional HhP inhibitor before, duringor after the other HhP inhibitor, or a combination of two or more of theforegoing. As indicated above, if one or more additional HhP inhibitorsare administered, it may be desirable for the additional HhPinhibitor(s) to differ from the currently administered HhP inhibitor(s)in its mechanism of action by which it inhibits the HhP (e.g.,itraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite of itraconazole, and vismodegib, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite of vismodegib). Multiple samples may be obtained andmeasurements determined and compared during the course of the treatmentto monitor the proliferation disorder over time.

An assessment of a subject's clinical response to HhP inhibition therapymay be made based on Hh levels or signaling, which may be assesseddirectly or indirectly by measuring a biomarker (an HhP biomarker) thatrepresents the HhP signal itself or a modulator of the HhP signal(inducer or inhibitor). If the biomarker is an inhibitor of the HhPsignal, and the level of the inhibitor is below normal, an assumptionmay be made that the HhP signal is elevated above normal. Likewise, ifthe biomarker is an inhibitor of the HhP signal, and the level of theinhibitor is above normal, an assumption may be made that the HhP signalis reduced below normal. If the biomarker is an inducer of the HhPsignal, and the level of the inducer is below normal, an assumption maybe made that the HhP signal is reduced below normal. Likewise, if thebiomarker is an inducer of the HhP signal, and the level of thebiomarker is above normal, an assumption may be made that the HhP signalis elevated above normal. Optionally, the accuracy of the aforementionedassumptions may be confirmed by measuring HhP signaling directly or bymeasuring other additional HhP biomarkers.

Hh levels or signaling may be monitored by measuring a biomarkerrepresentative of HhP activity, such as an Hh protein, or a nucleic acidencoding an HhP protein, such as an HhP ligand that activates thepathway and/or an upstream or downstream component(s) of the HhP, e.g.,a receptor, activator or inhibitor of hedgehog, is analyzed. Ligands ofthe mammalian HhP include Sonic hedgehog (SHH), desert hedgehog (DHH),and Indian hedgehog (DHH). The levels of Gli transcription factors maybe assessed as well (e.g., Gli1, Gli2, Gli3, or a combination or two ormore of the foregoing).

Any of the aforementioned biomarkers can be detected in a sampleobtained from the subject such as blood, urine, circulating tumor cells,a tumor biopsy, or a bone marrow biopsy. These biomarkers can also bedetected by systemic administration of a labeled form of an antibody toa biomarker followed by imaging with an appropriate imaging modality. Ifa biomarker representative of HhP activity is measured and when comparedto a reference level of that biomarker (a normal control or a levelmeasured in a sample obtained from the subject at an earlier time, suchas before initiation of the HhP inhibitor treatment), HhP signaling hasincreased or stayed the same following treatment with the HhP inhibitor,it can indicate a lack of clinical response to the treatment and a needto modify the treatment regimen by increasing the dose of the HhPinhibitor, or increasing the frequency of the dosing of the HhPinhibitor, or administering an additional HhP inhibitor before, duringor after the HhP inhibitor currently being administered, or acombination of two or more of the foregoing. As indicated above, if oneor more additional HhP inhibitors are administered, it may be desirablefor the additional HhP inhibitor(s) to differ from the first HhPinhibitor in its mechanism of action by which it inhibits the HhP (e.g.,itraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite of itraconazole, and vismodegib, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite of vismodegib). If a biomarker representative of HhP activityis measured (e.g., after about four weeks of administering the HhPinhibitor) and when compared to a reference level of that biomarker (anormal control or a level measured in a sample obtained from the subjectat an earlier time, such as before initiation of the HhP inhibitortreatment), relative reduction of HhP signaling indicates that the HhPinhibitor dose, the HhP inhibitor dose frequency, and the choice of HhPinhibitor(s) currently being administered are satisfactory and thetreatment may proceed in the absence of any adverse effects of thetreatment. The HhP inhibitor dose and/or frequency of dose may bereduced if any adverse effects are observed. Multiple samples may beobtained and measurements determined and compared during the course ofthe treatment to monitor the proliferation disorder over time. By way ofexample, if the proliferation disorder is basal cell carcinoma,monitoring may comprise measuring Gli1 in a sample of skin tissue ortumor taken at one or more time points following HhP inhibitoradministration (e.g., after about four weeks of administering the HhPinhibitor) and comparing the measured level of Gli1 to a reference level(a normal control or a level measured in a sample obtained from thesubject at an earlier time, such as before initiation of HhP inhibitortreatment). If Gli1 increases or stays the same following treatment withthe HhP inhibitor, it suggests a lack of clinical response to thetreatment and can indicate a need to modify the treatment regimen asindicated above, by increasing the dose of the HhP inhibitor, orincreasing the frequency of the dosing of the HhP inhibitor, oradministering an additional HhP inhibitor before, during or after theother HhP inhibitor, or a combination of two or more of the foregoing.Multiple samples may be obtained and measurements determined andcompared during the course of the treatment to monitor the proliferationdisorder over time.

Biomarker Detection

The methods of the invention may comprise assaying the presence, level,or activity of one or more biomarkers in a sample obtained from asubject before, during, and/or after administering the HhP inhibitor tothe subject. In some embodiments, the biomarker is associated with aproliferation disorder. For example, if the proliferation disorder is acancer, the biomarker may be a tumor-specific antigen ortumor-associated antigen. In some embodiments, the biomarker isassociated with a clinical response or lack thereof, such as the extentof HhP signaling. Examples of such biomarkers include Gli1, Gli2, Gli3,HhP ligand (such as Sonic hedgehog (SHH), desert hedgehog (DHH), orIndian hedgehog (DHH)), upstream or downstream component of the HhP(such as a receptor, activator, or inhibitor), PSA, and the plasma levelof an administered HhP inhibitor or its metabolite.

Optionally, it can be determined whether the biomarker level hassubsequently increased, diminished, or remained the same (e.g., incharacter and/or extent) relative to a reference biomarker level.

An assessment can be made of the subject's biomarker level one or moretimes after the initial treatment with the HhP inhibitor. Preferably, anassessment of the subject's biomarker level is also made before, during,or immediately after the subject's initial treatment with the HhPinhibitor (e.g., to establish a control or base-line for comparison to asubsequent assessment or assessments post-treatment). This may serve asa biomarker reference level. For example, an assessment of a biomarkerlevel can be made from a sample obtained from the subject beforetreatment with the HhP inhibitor but after treatment with one or moreother modalities such as chemotherapy, immunotherapy, and/or surgery.

In the methods of the invention, the subject's biomarker level can bemonitored by making multiple assessments after the initial treatment atuniform time intervals (e.g., daily, weekly, monthly, or annually) or atnon-uniform time intervals. Monitoring of the subject's biomarker levelcan continue for a pre-determined period of time, for a time determinedbased on therapeutic outcome, or indefinitely. Preferably, the subject'sbiomarker level is monitored from a time period starting prior toinitial treatment with the HhP inhibitor and continuing for a period oftime afterward (for example, for a period of at least five years), orindefinitely through the subject's life.

Typically, each assessment will involve obtaining an appropriatebiological sample from the subject. The appropriate biological samplemay depend upon the particular aspect of the subject's biomarker to beassessed (e.g., depending upon the particular assay). For example, insome embodiments, the biological sample will be one or more specimensselected from among whole blood, serum, peripheral blood mononuclearcells (PBMC), and a tissue (e.g., a tumor). Samples for assessments aretaken at a time point appropriate to obtain information regarding thebiomarker at the time of interest. For example, a sample may be takenfrom the subject from a time prior to administration of the HhPinhibitor and additional samples may be taken from the subjectperiodically after administration to determine the nature and extent ofthe biomarker levels observed.

The presence or level of biomarkers can be determined by measuring thelevel of biomarker nucleic acid (DNA or mRNA) or protein using knowntechniques. For example, immunological monitoring methods (i.e., animmunoassay) may be utilized to determine the level of biomarker, suchas a competitive or immunometric assay. The assay may be, for example, aradioimmunoassay (MA), immunoradiometric assay (IRMA), enzyme-linkedimmunosorbent assay (ELISA), dot blot, slot blot, enzyme-linkedimmunosorbent spot (ELISPOT) assay, Western blot, Northern blot,Southern blot, peptide microarray, or nucleic acid microarray. The levelof biomarker can be determined using surface plasmon resonance,fluorescence resonance energy transfer, bioluminescence resonance energytransfer, fluorescence quenching fluorescence, fluorescencepolarization, mass spectrometry (MS), high-performance liquidchromatography (HPLC), high-performance liquid chromatography/massspectrometry (HPLC/MS), high-performance liquid chromatography/massspectrometry/mass spectrometry (HPLC/MS/MS), capillary electrophoresis,rod-gel electrophoresis, or slab-gel electrophoresis. The level ofbiomarker can be determined using RT-PCR, PCR, nucleic acid sequencebased amplification assays (NASBA), transcription mediated amplification(TMA), or computerized detection matrix.

Assay standardization can include specific parameters to control forgeneral variability, such as assay conditions, sensitivity andspecificity of the assay, any in vitro amplification step involved,positive and negative controls, cutoff values for determining positiveand negative test results from subjects' samples, and any statisticalanalytical methods to be used for test results can be determined andselected by one of ordinary skill in the art.

A reference level of a biomarker that the determined biomarker level ofthe sample is compared against may be, for example, a level from asample obtained from the subject at an earlier time point (before orafter administration of the HhP inhibitor), or the reference level ofbiomarker may be a normal level or a statistically calculated level froman appropriate subject population, representing a level that isconsistent with a positive (desired) clinical outcome (i.e., the HhPinhibitor exhibits some degree of efficacy for the subject) or that isinconsistent with a positive clinical outcome (i.e., the HhP inhibitordoes not exhibit efficacy for the subject). The reference level may be asingle value (e.g., a cutoff value), a range, etc. For example, thereference level may be a range such that if the subject's biomarkerlevel does not reach the reference level or falls within the range, thesubject's biomarker level is deemed acceptable and no action need betaken. Conversely, if the subject's biomarker level reaches or exceedsthe reference level or falls outside the acceptable range, this canindicate that some action should be taken, such as withholding orceasing treatment with the HhP inhibitor, or reducing the amount of HhPinhibitor administered, and, optionally, administering an alternativetreatment, i.e., other than an HhP inhibitor.

Examples of biomarkers that can be determined or assayed includeprostate-specific antigen (PSA) in serum and PCA2 antigen in urine forprostate cancer. Another example of a biomarker that can be determinedor assayed is Gli in whole blood, serum, plasma, urine, cerebrospinalfluid, and tissue for a variety of proliferation disorders, includingcancers (see, for example, U.S. Patent Publication No. 20120083419,Altaba A. et al., “Methods and Compositions for InhibitingTumorigenesis,” the content of which is incorporated herein by referencein its entirety). Other examples of biomarkers that are associated withcancers (i.e., that are consistent with or correlate with cancer) can befound at www.cancer.gov/cancertopics/factsheet/detection/tumor-markers,including ALK gene rearrangements in tumors for non-small cell lungcancer and anaplastic large cell lymphoma, alpha-fetoprotein (AFP) inblood for liver cancer and germ cell tumors, beta-2-microglobulin (B2M)in blood, urine, or cerebrospinal fluid for multiple myeloma, chroniclymphocytic leukemia, and some lymphomas, beta-human chorionicgonadotropin (beta-hcG) in urine or blood for choriocarcinoma andtesticular cancer, BCR-ABL fusion gene in blood and/or bone marrow forchronic myeloid leukemia, BRAF mutation V600E in tumors for cutaneousmelanoma and colorectal cancer, CA15-3/CA27.29 in blood for breastcancer, CA19-9 in blood for pancreatic cancer, gallbladder cancer, bileduct cancer, and gastric cancer, CA-125 in blood for ovarian cancer,calcitonin in blood for medullary thyroid cancer, carcinoembryonicantigen (CEA) in blood for colorectal cancer and breast cancer, CD20 inblood for non-Hodgkin lymphoma, chromogranin A (CgA) in blood forneuroendocrine tumors, chromosomes 3, 7, 17, and 9p21 in urine forbladder cancer, cytokeratin fragments 21-1 in blood for lung cancer,CGFR mutation analysis in tumors for non-small cell lung cancer,estrogen receptor (ER)/progesterone receptor (PR) in tumors for breastcancer, fibrin/fibrinogen in urine for bladder cancer, HE4 in blood forovarian cancer, HER2/neu in tumors for breast cancer, gastric cancer,and esophageal cancer, immunoglobulins in blood and urine for multiplemyeloma and Waldenstrom macroglobulinemia, KIT in tumors forgastrointestinal stromal tumor and mucosal melanoma, KRAS mutationanalysis in tumors for colorectal cancer and non-small cell lung cancer,lactate dehydrogenase in blood for germ cell tumors, nuclear matrixprotein 22 in urine for bladder cancer, thyroglobulin in tumors forthyroid cancer, urokinase plasminogen activator (uPA) and plasminogenactivator inhibitor (PAI-1) in tumors for breast cancer, 5-proteinsignature (Oval) in blood for ovarian cancer, 21-gene signature(oncotype DX) in tumors for breast cancer, and 70-gene signature(mammaprint) cancer.gov/cancertopics/factsheet/detection/tumor-markers.

In some embodiments, the biomarker comprises PSA. PSA, also known asgamma-seminoprotein or kallikrein-3 (KLK3), is a glycoprotein enzymeencoded in humans by the KLK3 gene. PSA is a member of thekallikrein-related peptidase family. In the methods of the invention,determination or measurement of PSA level in a sample may be madedirectly by assessment of the amount of nucleic acid (e.g., DNA or mRNA)encoding PSA, PSA polypeptide (PSA gene product), or in the activity ofPSA. Examples of PSA measurement methods that may be utilized includebut are not limited to those described in Blase A. B. et al., “Five PSAMethods Compared by Assaying Samples with Defined PSA Ratios,” ClinicalChemistry, May 1997, 43(5):843-845; Gelmini S. et al., “Real-time RT-PCTFor The Measurement of Prostate-Specific Antigen mRNA Expression inBenign Hyperplasia and Adenocarcinoma of Prostate,” Clin. Chem. Lab.Med., 2003 March, 41(3):261-265; and Kalfazade N. et al.,“Quantification of PSA mRNA Levels in Peripheral Blood of Patients withLocalized Prostate Adenocarcinoma Before, During and After RadicalProstatectomy by Quantitative Real-Time PCR (qRT-PCR),” Int. Urol.,Nephrol., 2009, Epub 2008 Jun. 27, 41(2):273-279, which are eachincorporated herein by reference in its entirety.

In accordance with the invention, PSA level may be determined bymeasuring total PSA (tPSA; measure of all PSA in a sample), free PSA(fPSA; amount free, unbound PSA protein), or complex PSA (cPSA; theamount of PSA that is complexed with or bound to other proteins) in asample. Optionally, determination of PSA level further comprisesdetermining PSA velocity or PSA doubling time. PSA velocity is the rateof change in a subject's PSA level over time, typically expressed asng/mL per year. PSA doubling time is the period of time over which asubject's PSA level doubles. Pro-PSA refers to several differentinactive precursors of PSA. Preferably, the mature, active form of PSA,lacking the leader peptide, is determined. However, pro-PSA may bemeasured as an alternative, or in addition to, the mature form (MasoodA. K. et al., “Evolving Role of Pro-PSA as a New Serum Marker for theEarly Detection of Prostate Cancer”, Rev. Urol., 2002, 4(4):198-200).

The methods of the invention may comprise assessing the level of PSA ina sample obtained from a subject before, during, and/or afteradministering the HhP inhibitor to the subject to determine whether thePSA level has subsequently increased, diminished, or remained the same(e.g., in character and/or extent) relative to a reference PSA level.

An assessment can be made of the subject's PSA level one or more timesafter the initial treatment with the HhP inhibitor. Preferably, anassessment of the subject's PSA level is also made before, during, orimmediately after the subject's initial treatment with the HhP inhibitor(e.g., to establish a control or base-line for comparison to asubsequent assessment or assessments post-treatment). This may serve asa PSA reference level. For example, an assessment of PSA level can bemade from a sample obtained from the subject before treatment with theHhP inhibitor but after treatment with one or more other modalities suchas chemotherapy, immunotherapy, and/or surgery.

In the methods of the invention, the subject's PSA level can bemonitored by making multiple assessments after the initial treatment atuniform time intervals (e.g., daily, weekly, monthly, or annually) or atnon-uniform time intervals. Monitoring of the subject's PSA level cancontinue for a pre-determined period of time, for a time determinedbased on therapeutic outcome, or indefinitely. Preferably, the subject'sPSA level is monitored from a time period starting prior to initialtreatment with the HhP inhibitor and continuing for a period of timeafterward (for example, for a period of at least five years), orindefinitely through the subject's life.

Typically, each assessment will involve obtaining an appropriatebiological sample from the subject. The appropriate biological samplemay depend upon the particular aspect of the subject's PSA to beassessed (e.g., depending upon the particular assay). For example, insome embodiments, the biological sample will be one or more specimensselected from among whole blood, serum, peripheral blood mononuclearcells (PBMC), and a tissue (e.g., a tumor). Samples for assessments aretaken at a time point appropriate to obtain information regarding thePSA at the time of interest. For example, a sample may be taken from thesubject from a time prior to administration of the HhP inhibitor andadditional samples may be taken from the subject periodically afteradministration to determine the nature and extent of the PSA levelsobserved.

The level of PSA can be determined by measuring the level of PSA nucleicacid (DNA or mRNA) or protein using known techniques. For example,immunological monitoring methods (i.e., an immunoassay) may be utilizedto determine the level of PSA, such as a competitive or immunometricassay. The assay may be, for example, a radioimmunoassay (MA),immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay(ELISA), dot blot, slot blot, enzyme-linked immunosorbent spot (ELISPOT)assay, Western blot, Northern blot, Southern blot, peptide microarray,or nucleic acid microarray. The level of PSA can be determined usingsurface plasmon resonance, fluorescence resonance energy transfer,bioluminescence resonance energy transfer, fluorescence quenchingfluorescence, fluorescence polarization, mass spectrometry (MS),high-performance liquid chromatography (HPLC), high-performance liquidchromatography/mass spectrometry (HPLC/MS), high-performance liquidchromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS),capillary electrophoresis, rod-gel electrophoresis, or slab-gelelectrophoresis. The level of PSA can be determined using RT-PCR, PCR,nucleic acid sequence based amplification assays (NASBA), transcriptionmediated amplification (TMA), or computerized detection matrix.

Assay standardization can include specific parameters to control forgeneral variability, such as assay conditions, sensitivity andspecificity of the assay, any in vitro amplification step involved,positive and negative controls, cutoff values for determining positiveand negative test results from subjects' samples, and any statisticalanalytical methods to be used for test results can be determined andselected by one of ordinary skill in the art.

A reference level of PSA that the determined PSA level of the sample iscompared against may be, for example, a level from a sample obtainedfrom the subject at an earlier time point (before or afteradministration of the HhP inhibitor), or the reference level of PSA maybe a statistically calculated level from an appropriate subjectpopulation, representing a level that is consistent with a positive(desired) clinical outcome (i.e., the HhP inhibitor exhibits some degreeof efficacy for the subject) or that is inconsistent with a positiveclinical outcome (i.e., the HhP inhibitor does not exhibit efficacy forthe subject). The reference level may be a single value (e.g., a cutoffvalue), a range, etc. For example, the reference level may be a rangesuch that if the subject's PSA level does not reach the reference levelor falls within the range, the subject's PSA level is deemed acceptableand no action need be taken. Conversely, if the subject's PSA levelreaches or exceeds the reference level or falls outside the acceptablerange, this can indicate that some action should be taken, such aswithholding or ceasing treatment with the HhP inhibitor, or reducing theamount of HhP inhibitor administered, and, optionally, administering analternative treatment, i.e., other than an HhP inhibitor.

The methods of the invention can further include the step of monitoringthe subject, e.g., for a change (e.g., an increase or decrease) in oneor more of: tumor size; hedgehog levels or signaling; stromalactivation; levels of one or more cancer markers; the rate of appearanceof new lesions; the appearance of new disease-related symptoms; the sizeof soft tissue mass, e.g., a decreased or stabilization; quality oflife, e.g., amount of disease associated pain; or any other parameterrelated to clinical outcome. The subject can be monitored in one or moreof the following periods: prior to beginning of treatment; during thetreatment; or after one or more elements of the treatment have beenadministered. Monitoring can be used to evaluate the need for furthertreatment with the same HhP inhibitor, alone or in combination with, thesame therapeutic agent, or for additional treatment with additionalagents. Generally, a decrease in one or more of the parameters describedabove is indicative of the improved condition of the subject, althoughwith serum hemoglobin levels, an increase can be associated with theimproved condition of the subject.

The methods of the invention can further include the step of analyzing anucleic acid or protein from the subject, e.g., analyzing the genotypeof the subject. In one embodiment, a hedgehog protein, or a nucleic acidencoding a hedgehog ligand and/or an upstream or downstream component(s)of the hedgehog signaling, e.g., a receptor, activator or inhibitor ofhedgehog, is analyzed. The elevated hedgehog ligand can be detected inblood, urine, circulating tumor cells, a tumor biopsy or a bone marrowbiopsy. The elevated hedgehog ligand can also be detected by systemicadministration of a labeled form of an antibody to a hedgehog ligandfollowed by imaging. In addition to determination of PSA in accordancewith the invention, the analysis can be used, e.g., to evaluate thesuitability of, or to choose between alternative treatments, e.g., aparticular dosage, mode of delivery, time of delivery, inclusion ofadjunctive therapy, e.g., administration in combination with a secondagent, or generally to determine the subject's probable drug responsephenotype or genotype. The nucleic acid or protein can be analyzed atany stage of treatment, but preferably, prior to administration of theHhP inhibitor and/or therapeutic agent, to thereby determine appropriatedosage(s) and treatment regimen(s) of the HhP inhibitor (e.g., amountper treatment or frequency of treatments) for prophylactic ortherapeutic treatment of the subject.

In certain embodiments, the methods of the invention further include thestep of detecting elevated hedgehog ligand in the subject, prior to, orafter, administering a HhP inhibitor to the subject. The elevatedhedgehog ligand can be detected in blood, urine, circulating tumorcells, a tumor biopsy or a bone marrow biopsy. The elevated hedgehogligand can also be detected by systemic administration of a labeled formof an antibody to a hedgehog ligand followed by imaging. The step ofdetecting elevated hedgehog ligand can include the steps of measuringhedgehog ligand in the patient prior to administration of the othercancer therapy, measuring hedgehog ligand in the patient afteradministration of the other cancer therapy, and determining if theamount of hedgehog ligand after administration of the other chemotherapyis greater than the amount of hedgehog ligand before administration ofthe other chemotherapy. The other cancer therapy can be, for example, atherapeutic agent or radiation therapy.

Hedgehog Pathway Signaling Inhibitors

Hh pathway activation begins when the Hh ligand binds to and inhibitsthe transmembrane receptor Patched1 (Ptch1), allowing the signaltransducer Smoothened (Smo) to activate Gli transcription factors andamplify Hh target gene expression. Thus far, all of the nuclear eventsascribed to Hh occur through the Gli transcription factors, with Gli1acting predominantly as an activator, Gli3 acting predominantly as arepressor, and Gli2 possessing both repressive and activator functions.

Any HhP inhibitor may be used in the invention as a monotherapy or incombination regimens with one or more other HhP inhibitors and/or incombination with one or more other therapeutic or prophylactic agents ortreatments, such as chemotherapeutic agents, radiation, surgery, andimmunotherapy. HhP inhibitors and biological assays and in vivo modelsthat may be employed for the identification and characterization ofinhibitors of various members of the HhP are described in Peukert S. andMiller-Moslin K., “Small-Molecule Inhibitors of the Hedgehog SignalingPathway as Cancer Therapeutics”, Chem Med Chem, 2010, 5(4):500-512,Sahebjam, et al., “The Utility of Hedgehog Signaling Pathway Inhibitionfor Cancer”, The Oncologist, 2012, 17:1090-1099; Liu H. et al.,“Clinical Implications of Hedgehog Signaling Pathway Inhibitors,” Chin.J. Cancer, 2011, 30(1):13-26; Atwood Scott X. et al., “Hedgehog PathwayInhibition and the Race Against Tumor Evolution,” J. Cell Biol.,199(2):193-197; and U.S. Patent Publication No. 20090203713, Beachy P.A. et al., “Hedgehog Pathway Antagonists to Treat Disease,” the contentsof each of which is incorporated herein by reference in its entirety.

Drug discovery efforts aimed at identifying inhibitors of the Hhsignaling pathway have facilitated the development of a multitude ofbiological assay systems for interrogating Hh pathway activity,including cell-based assays, tissue assays, and at least one in vivoassay, and binding assays have been used to confirm the specificproteins in the pathway being targeted. In addition, animal diseasemodels have been established for a variety of cancer types, includingmedulloblastoma, basal cell carcinoma (BCC), breast cancer, lymphoma,and chronic myeloid leukemia (CIVIL), as well as pancreatic, prostate,colorectal and small-cell lung cancer (SCLC). These models have beenused to evaluate the effects of various small molecule HhP inhibitors ontumor growth and progression.

The Smoothened receptor (Smo) has thus far shown to be the most“druggable” target in the pathway, as demonstrated by the structurallydiverse array of both naturally occurring and fully synthetic smallmolecule Smo inhibitors reported. Efforts are ongoing to identifyadditional druggable nodes in the pathway, and promising initial resultshave been demonstrated for targeting the Sonic hedgehog protein (Shh)and the downstream target Gli1 with small molecule inhibitors.

The most common way to target HhP is modulation of Smo. Smo is a Gprotein-coupled receptor protein encoded by the Smo gene of the HhP. Smois the molecular target of the teratogen cyclopamine. Antagonists andagonists of Smo have been shown to affect the pathway regulationdownstream. The most clinically advanced Smo targeting agents arecyclopamine-competitive. Itraconazole (Sporanox) has also been shown totarget Smo through a mechanism distinct from cyclopamine and vismodegib.Itraconazole inhibits Smo in the presence of mutations conferringresistance to vismodegib and other cyclopamine-competitive antagonistssuch as IPI-926 and LDE-225. Ptch and Gli3 (5E1) antibodies are also away to regulate the pathway. A downstream effector and strongtranscriptional activator siRNA Gli1 has been used to inhibit cellgrowth and promote apoptosis. Arsenic trioxide (Trisenox) has also beenshown to inhibit hedgehog signaling by interfering with Gli function andtranscription.

As used herein, the terms “hedgehog inhibitor”, “hedgehog pathwayinhibitor”, “HhP inhibitor”, or in most contexts “inhibitor” refer to anagent capable of blocking or reducing cellular responses to the hedgehogsignaling pathway, e.g., in cells with an active hedgehog signalingpathway, and more specifically, inhibiting cellular responses, directlyor indirectly, to the hedgehog family of secreted growth factors. Thehedgehog inhibitor may antagonize hedgehog pathway activity through anumber of routes, including, but not limited to, by interfering with theinhibitory effect that Ptch exerts on Smo; by activating Smo withoutaffecting Ptc; by influencing Smo function by directly binding to Smo;and/or by activating the pathway downstream of Smo. Exemplary hedgehoginhibitors may include, but are not limited to, steroidal alkaloids suchas cyclopamine and jervine. In some embodiments, the HhP inhibitorantagonizes HhP activity by binding to a component (effector molecule)of the pathway (e.g., a Hedgehog receptor such as Ptch or Smo, or asignaling mediator such as Gli1, Gli2, or Gli3), interfering with theinhibitory effect that a component of the pathway exerts on anothercomponent of the pathway, by activating a component of the pathwaywithout affecting another component, by activating a component of thepathway downstream of Smo, or by reducing or eliminating expression of acomponent of the pathway. In some embodiments, the HhP inhibitorantagonizes HhP activity by binding to Smo, interfering with theinhibitory effect that Ptch exerts on Smo, by activating Smo withoutaffecting Ptch, by activating the pathway downstream of Smo, or byreducing or eliminating expression of Smo. In some embodiments, the HhPinhibitor is cyclopamine-competitive. In some embodiments, the HhPinhibitor is cyclopamine-competitive and the proliferation disorder islung cancer, basal cell carcinoma, prostate cancer, or other cancer. TheHhP inhibitor may be any class of agent or treatment capable of blockingor reducing cellular responses to the HhP and may be, for example, apolypeptide (e.g., protein, peptide, immunoglobulin (antibody orantibody fragment)), a nucleic acid (e.g., antisense molecule, ribozyme,or interfering RNA such as siRNA or shRNA), or a small molecule. The HhPinhibitor may be active upon administration to the subject, and/oractive upon metabolic processing or other mechanisms in vivo (i.e., asone or more active metabolites).

Although the term “HhP inhibitor” and its grammatical variants are usedherein to refer to agents capable of blocking or reducing cellularresponses to the hedgehog signaling pathway, e.g., in cells with anactive hedgehog signaling pathway, and more specifically, inhibitingcellular responses, directly or indirectly, to the hedgehog family ofsecreted growth factors, the invention encompasses use of HhP inhibitorsto treat proliferation disorders (e.g., cancer), whether that particularagent's primary mechanism of action in treating the proliferationdisorder in question is through the above-described HhP inhibition orthrough some other mechanism of action, such as inhibition ofangiogenesis. For example, itraconazole is an HhP inhibitor and inhibitsangiogenesis. In treating some cancers in accordance with the invention,the HhP inhibitor may act by a mechanism completely independent of itsHhP inhibition properties. Thus, the identification of an agent as beingan HhP inhibitor is not limited to the context in which it is beingused, but rather to its ability to inhibit the HhP.

Suitable hedgehog inhibitors for use with the present invention include,for example, those described and disclosed in U.S. Pat. No. 7,230,004,U.S. Patent Application Publication No. 2008/0293754, U.S. PatentApplication Publication No. 2008/0287420, U.S. Patent ApplicationPublication No. 2008/0293755, and U.S. Patent Application PublicationNo. 2008/0019961, the entire disclosures of which are incorporated byreference herein.

Examples of other suitable hedgehog inhibitors also include thosedescribed in U.S. Patent Application Publication Nos. US 2002/0006931,US 2007/0021493 and US 2007/0060546, and International ApplicationPublication Nos. WO 2001/19800, WO 2001/26644, WO 2001/27135, WO2001/49279, WO 2001/74344, WO 2003/011219, WO 2003/088970, WO2004/020599, WO 2005/013800, WO 2005/033288, WO 2005/032343, WO2005/042700, WO 2006/028958, WO 2006/050351, WO 2006/078283, WO2007/054623, WO 2007/059157, WO 2007/120827, WO 2007/131201, WO2008/070357, WO 2008/110611, WO 2008/112913, and WO 2008/131354.

Additional examples of HhP inhibitors include, but are not limited to,GDC-0449 (also known as RG3616 or vismodegib) described in, e.g., VonHoff D. et al., N. Engl. J. Med. 2009; 361(12):1164-72; Robarge K. D. etal., Bioorg. Med. Chem. Lett., 2009; 19(19):5576-81; Yauch, R. L. etal., Science, 2009, 326: 572-574; Rudin, C. et al., New England J ofMedicine, 2009, 361-366; BMS-833923 (also known as XL139) described in,e.g., in Siu L. et al., J. Clin. Oncol. 2010; 28:15s (suppl; abstr2501); and National Institute of Health Clinical Trial Identifier No.NCT006701891; LDE-225 described, e.g., in Pan S. et al., ACS Med. Chem.Lett., 2010; 1(3): 130-134; LEQ-506 described, e.g., in NationalInstitute of Health Clinical Trial Identifier No. NCT01106508;PF-04449913 described, e.g., in National Institute of Health ClinicalTrial Identifier No. NCT00953758; Hedgehog pathway antagonists disclosedin U.S. Patent Application Publication No. 2010/0286114; SMOi2-17described, e.g., U.S. Patent Application Publication No. 2010/0093625;SANT-1 and SANT-2 described, e.g., in Rominger C. M. et al., J.Pharmacol. Exp. Ther., 2009; 329(3):995-1005;1-piperazinyl-4-arylphthalazines or analogues thereof, described inLucas B. S. et al., Bioorg. Med. Chem. Lett., 2010; 20(12):3618-22.

HhP inhibitors useful in the current invention can contain a basicfunctional group, such as amino or alkylamino, and are thus capable offorming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately treating thecompound in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed during subsequent purification.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate, besylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like(see, for example, Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci.,1977, 66:1-19).

Pharmaceutically acceptable salts include, but are not limited to,conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include, but are not limited to, thosederived from inorganic acids such as hydrochloride, hydrobromic,sulfuric, sulfamic, phosphoric, nitric, and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,benzenesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the HhP inhibitors can contain one or more acidicfunctional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately treating the compound in itsfree acid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically-acceptable metal cation, with ammonia,or with a pharmaceutically-acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

If administered with another therapeutic agent, the HhP inhibitor andthe therapeutic agent can be administered as separate compositions,e.g., pharmaceutical compositions, or administered separately, but viathe same route (e.g., both orally or both intravenously), oradministered in the same composition, e.g., pharmaceutical composition.

In one embodiment, the HhP inhibitor is administered prior to detectionof the proliferation disorder. In another embodiment, the HhP inhibitoris administered after detection of the proliferation disorder. In oneembodiment, the proliferation disorder is cancer (prostate cancer, basalcell carcinoma, lung cancer, or other cancer), and the HhP inhibitor isadministered prior to detection of the cancer. In another embodiment,the proliferation disorder is cancer (prostate cancer, basal cellcarcinoma, lung cancer, or other cancer), and the HhP inhibitor isadministered after detection of the cancer.

Some HhP inhibitors may comprise one or more asymmetric centers, andthus can exist in various isomeric forms, i.e., stereoisomers(enantiomers, diastereomers, cis-trans isomers, E/Z isomers, etc.).Thus, HhP inhibitors can be in the form of an individual enantiomer,diastereomer or other geometric isomer, or can be in the form of amixture of stereoisomers. Enantiomers, diastereomers and other geometricisomers can be isolated from mixtures (including racemic mixtures) byany method known to those skilled in the art, including chiral highpressure liquid chromatography (HPLC) and the formation andcrystallization of chiral salts or prepared by asymmetric syntheses;see, for example, Jacques, et al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron, 1977, 33:2725; Eliel, E. L. Stereochemistry of CarbonCompounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind. 1972).

Hedgehog pathway inhibitors are exemplified herein by itraconazole,including pharmaceutically acceptable, salts, prodrugs, isomers, andmetabolites thereof. Isomers of itraconazole include each of itsstereoisomers (Castro-Puyana M. et al., “Separation and Quantitation ofthe Four Stereoismers of Itraconazole in Pharmaceutical Formulations byElectrokinetic Chromatography”, Electrophoresis, 2006, 27(4):887-895;Kunze K. L. et al., “Stereochemical Aspects of Itraconazole MetabolismIn Vitro and In Vivo,” Drug Metab. Dispos., 2006, Epub 2006 Jan. 13,34(4):583-590, and as corrected in “Correction to “StereochemicalAspects of Itraconazole Metabolism In Vitro and In Vivo,” Drug Metab.Dispos., 2012, 40(12):2381); Chong C. R. et al., “Inhibition ofAngiogenesis by the Antifungal Drug Itraconazole,” ACS Chemical Biology,2007, 2(4):263-270; Kim J. et al., “Itraconazole, a Commonly UsedAntifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth,”Cancer Cell, 2010, 17(4):388-399); Patent Publication No.WO/2008/124132, Liu J. et al., entitled “Chirally Pure Isomers ofItraconazole and Inhibitors of Lanosterol 14A-Demethylase For Use asAngiogenesis Inhibitors”). In some embodiments, the HhP inhibitorcomprises a stereoisomer of itraconazole selected from (2R,4S,2′R),(2R,4S,2′S), (2S,4R,2S′R), or (2S,4R2′S). In some embodiments, the HhPinhibitor comprises an itraconazole analogue in which the sec-butyl sidechain has been replaced with one or more moieties, relative toitraconazole. For example, the itraconazole analogue may be one in whichthe native sec-butyl side chain is replaced with C₁-C₈ alkyl, C₂-C₈alkenyl, or C₂-C₈ alkynyl, that are straight, branched, or cyclic, andare unsubstituted or substituted one or more times at any position witha C₁-C₈ alkoxy, C₆-C₁₀ aryl, N₃, OH, Cl, Br, I, F, C₆-C₁₀ aryl oxy,C₁-C₈ alkyl carboxy, aryl carboxy, wherein any substituent can befurther substituted with any of the foregoing.

In some embodiments, the HhP inhibitor is an azole antifungaldrug-containing composition as described in U.S. Patent ApplicationPublication No. 20030225104 (Hayes et al., “Pharmaceutical Compositionsfor Poorly Soluble Drugs,” issued as U.S. Pat. No. 6,881,745 which isincorporated herein by reference in its entirety). In some embodiments,the composition in vivo provides a mean C_(MAX) of at least about 100ng/ml (e.g., 150 to 250 ng/ml) after administration in the fasted state.In some embodiments, the HhP inhibitor is a composition including anazole antifungal drug, such as itraconazole, and at least one polymerhaving one or more acidic functional groups. In a particularly preferredform the polymer is a polycarboxylic acid such as a hydroxypropylmethylcellulose phthalate such as that available from Shin-Etsu ChemicalIndustry Co Ltd as HP-50, HP-55 or HP-55S. In some embodiments, the HhPinhibitor is a composition including an azole antifungal drug, such asitraconazole, and at least one polymer having one or more acidicfunctional groups, wherein the composition in vivo provides a meanC_(MAX) of at least 100 ng/ml (e.g., 150 to 250 ng/ml). In someembodiments, the HhP inhibitor is a composition including about 100 mgof an azole antifungal drug, such as itraconazole, and optionally atleast one polymer having acidic functional groups.

In some embodiments, the HhP inhibitor is the SUBACAP™ formulation ofitraconazole. The SUBACAP™ formulation is a solid dispersion whereinitraconazole is associated with acidic molecules and the formulationallows for excellent absorption at pH 5.5-7. Itraconazole release occursin the intestines; therefore, fed or fasted state does not affect theabsorption, nor are there restrictions for achlorhydric patients orpatients on proton-pump inhibitor drugs for high acid control.

In some embodiments, an HhP inhibitor such as itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, is administered in a SUBA® formulation at a dose inthe range of 100 mg to 600 mg per day. In some embodiments, 150 mg of anHhP inhibitor such as itraconazole, or a pharmaceutically acceptablesalt, prodrug, stereoisomer, or active metabolite thereof, isadministered in a SUBA® formulation two or more times per day. In someembodiments, 200 mg of an HhP inhibitor such as itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, is administered in a SUBA® formulation two or moretimes per day.

Methods of Treatment

One aspect of the invention concerns a method for treating aproliferation disorder in a subject, comprising administering acomposition comprising a Hedgehog pathway (HhP) inhibitor to thesubject, wherein the composition is administered (preferably, orally) inan effective amount to achieve a plasma trough level of at least about1,000 ng/mL of the HhP inhibitor.

The method of prognosticating an outcome of prostate cancer treatmentand method of determining the efficacy of HhP inhibitor therapy mayfurther comprise administering an HhP inhibitor therapy. Thus, anotheraspect of the invention concerns a method for treating prostate cancerin a subject, comprising administering Hedgehog pathway (HhP) inhibitortherapy to the subject; and carrying out a method of the invention(i.e., a method of prognosticating an outcome of prostate cancertreatment with a HhP inhibitor therapy, or a method of determining theefficacy of HhP inhibitor therapy).

In treating a proliferation disorder (e.g., prostate cancer, basal cellcarcinoma, lung cancer, or other cancer) one or more HhP inhibitors (andcompositions containing them) may be administered by any route effectivefor delivery to the desired tissues, e.g., administered orally,parenterally (e.g., intravenously), intramuscularly, sublingually,buccally, rectally, intranasally, intrabronchially, intrapulmonarily,intraperitoneally, topically, transdermally and subcutaneously, forexample. The HhP inhibitors can be formulated for the most effectiveroute of administration. For example, an HhP inhibitor may beadministered orally or locally (e.g., by direct injection) to a desiredsite, such as a precancerous lesion or tumor (e.g., prostate cancerlesion or prostate tumor or other cancer tumor). The amount administeredin a single dose may be dependent on the subject being treated, thesubject's weight, the manner of administration and the judgment of theprescribing physician. Generally, however, administration and dosage andthe duration of time for which a composition is administered willapproximate those which are necessary to achieve a desired result.

The selected dosage level of the HhP inhibitor will depend upon avariety of factors including, for example, the activity of theparticular compound employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

In general, a suitable daily dose of an HhP inhibitor will be thatamount of the inhibitor which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, oral, intravenous andsubcutaneous doses of the HhP inhibitor for a subject, when used for theindicated effects, will range from about 0.0001 mg to about 1000 mg perday, or about 0.001 mg to about 1000 mg per day, or about 0.01 mg toabout 1000 mg per day, or about 0.1 mg to about 1000 mg per day, orabout 0.0001 mg to about 600 mg per day, or about 0.001 mg to about 600mg per day, or about 0.01 mg to about 600 mg per day, or about 0.1 mg toabout 600 mg per day, or about 200 mg to 600 mg per day. The optimalpharmaceutical formulations can be readily determined by one of ordinaryskill in the art depending upon the route of administration and desireddosage. (See, for example, Remington's Pharmaceutical Sciences, 18th Ed.(1990), Mack Publishing Co., Easton, Pa., the entire disclosure of whichis hereby incorporated by reference).

The subject receiving treatment is any animal in need, includingprimates, in particular humans, equines, cattle, swine, sheep, poultry,dogs, cats, mice and rats. The subject may be any gender, though someproliferation disorders are gender-specific (e.g., prostate cancer,ovarian cancer).

The HhP inhibitors can be administered daily, every other day, threetimes a week, twice a week, weekly, or bi-weekly. The dosing schedulecan include a “drug holiday,” i.e., the drug can be administered for twoweeks on, one week off, or three weeks on, one week off, or four weekson, one week off, etc., or continuously, without a drug holiday. The HhPinhibitors can be administered orally, intravenously, intraperitoneally,topically, transdermally, intramuscularly, subcutaneously, intranasally,sublingually, or by any other route.

Single or multiple administrations of the HhP inhibitor can be carriedout with dose levels and patterns being selected by the treatingphysician, optionally based on the level of a biomarker (e.g., PSA levelfor prostate cancer) determined in a sample obtained from the subjectrelative to a reference biomarker level (e.g., reference PSA level).

In some embodiments, the HhP inhibitor is administered with one or moreother therapeutic treatments before, during, or after the HhP inhibitor.The HhP inhibitor and the therapeutic agent that is a non-HhP inhibitorcan be administered within the same formulation or differentformulations. If administered in different formulations, the HhPinhibitor and the therapeutic agent can be administered by the sameroute or by different routes.

Depending on the intended mode of administration, the inhibitors andtherapeutic agents used in the methods described herein may be in theform of solid, semi-solid or liquid dosage forms, such as, for example,tablets, suppositories, pills, capsules, powders, liquids, suspensions,lotions, creams, gels, or the like, preferably in unit dosage formsuitable for single administration of a precise dosage. Each dose mayinclude an effective amount of a compound used in the methods describedherein in combination with a pharmaceutically acceptable carrier and, inaddition, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, diluents, etc.

Liquid pharmaceutically administrable compositions can prepared, forexample, by dissolving, dispersing, etc., a compound for use in themethods described herein and optional pharmaceutical adjuvants in anexcipient, such as, for example, water, saline aqueous dextrose,glycerol, ethanol, and the like, to thereby form a solution orsuspension. For solid compositions, conventional nontoxic solid carriersinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose,sucrose, magnesium carbonate, and the like. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack PublishingCo., Easton, Pa., the entire disclosure of which is hereby incorporatedby reference).

Formulations comprising HhP inhibitors may be presented in unit-dose ormulti-dose containers (packs), for example, sealed ampoules and vials,and may be stored in a freeze dried (lyophilized) condition requiringonly the condition of the sterile liquid carrier, for example, water forinjections, prior to use. Examples of pack types that may be utilizedinclude, but are not limited to, multidose packs (also referred to asreclosables), such as bottles, aerosol packs, and tubes, and unit dosepacks (also referred to as non-reclosables), such as ampoules, blisterpacks pre-filled syringes, vials, sachets, and form/blow-fill-seal (FFS,BFS) in various pack formats. In one embodiment, the itraconazole is ina SUBA® formulation (e.g., SUBACAP™ formulation) presented in a blisterpack. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powder, granules, tablets, etc. It should be understoodthat in addition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

Patients in need of treatment using the methods and compositions of thepresent invention can be identified using standard techniques known tothose in the medical or veterinary professions, as appropriate. In someembodiments, the proliferation disorder to be treated is onecharacterized by upregulation (elevation) of Hh level and/or HhPsignaling above the constitutive level (or normal level for the normalcell type in question). As indicated above, optionally, subjects in needof treatment (or further treatment) of a proliferation disorder such asprostate cancer, basal cell carcinoma, lung cancer, or other cancer, maybe selected as an individual particularly suitable for treatment with anHhP inhibitor, based on Hh level or signaling, which may be assesseddirectly or indirectly by measuring a biomarker (an HhP biomarker) thatrepresents the HhP signal itself or a modulator of the HhP signal(inducer or inhibitor).

Cancer is an example of a proliferation disorder that may be treated andmonitored using methods of the invention. The terms “cancer” and“malignancy” are used herein interchangeably to refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. The methods and compositions of the inventioncan be utilized for early, middle, or late stage disease, and acute orchronic disease. The cancer may be drug-resistant or drug-sensitive.Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include breast cancer, prostate cancer, colon cancer, squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, cervical cancer, ovariancancer, peritoneal cancer, liver cancer, e.g., hepatic carcinoma,bladder cancer, colorectal cancer, endometrial carcinoma, kidney cancer,and thyroid cancer. In some embodiments, the cancer is a hematologicmalignancy (for example, multiple myeloma or leukemia). In someembodiments, the cancer is a non-hematologic malignancy.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer ofthe respiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; uterine cancer; cancer of the urinary system, as well as othercarcinomas and sarcomas. Examples of cancer types that may potentiallybe treated using the methods and compositions of the present inventionare also listed in Table 1.

TABLE 1 Examples of Cancer Types Acute Lymphoblastic Leukemia, AdultAcute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, AdultAcute Myeloid Leukemia, Childhood Adrenocortical CarcinomaAdrenocortical Carcinoma, Childhood AIDS-Related Cancers AIDS-RelatedLymphoma Anal Cancer Astrocytoma, Childhood Cerebellar Astrocytoma,Childhood Cerebral Basal Cell Carcinoma Bile Duct Cancer, ExtrahepaticBladder Cancer Bladder Cancer, Childhood Bone Cancer,Osteosarcoma/Malignant Fibrous Histiocytoma Brain Stem Glioma, ChildhoodBrain Tumor, Adult Brain Tumor, Brain Stem Glioma, Childhood BrainTumor, Cerebellar Astrocytoma, Childhood Brain Tumor, CerebralAstrocytoma/Malignant Glioma, Childhood Brain Tumor, Ependymoma,Childhood Brain Tumor, Medulloblastoma, Childhood Brain Tumor,Supratentorial Primitive Neuroectodermal Tumors, Childhood Brain Tumor,Visual Pathway and Hypothalamic Glioma, Childhood Brain Tumor, ChildhoodBreast Cancer Breast Cancer, Childhood Breast Cancer, Male BronchialAdenomas/Carcinoids, Childhood Burkitt' s Lymphoma Carcinoid Tumor,Childhood Carcinoid Tumor, Gastrointestinal Carcinoma of Unknown PrimaryCentral Nervous System Lymphoma, Primary Cerebellar Astrocytoma,Childhood Cerebral Astrocytoma/Malignant Glioma, Childhood CervicalCancer Childhood Cancers Chronic Lymphocytic Leukemia ChronicMyelogenous Leukemia Chronic Myeloproliferative Disorders Colon CancerColorectal Cancer, Childhood Cutaneous T-Cell Lymphoma, see MycosisFungoides and Sézary Syndrome Endometrial Cancer Ependymoma, ChildhoodEsophageal Cancer Esophageal Cancer, Childhood Ewing's Family of TumorsExtracranial Germ Cell Tumor, Childhood Extragonadal Germ Cell TumorExtrahepatic Bile Duct Cancer Eye Cancer, Intraocular Melanoma EyeCancer, Retinoblastoma Gallbladder Cancer Gastric (Stomach) CancerGastric (Stomach) Cancer, Childhood Gastrointestinal Carcinoid TumorGerm Cell Tumor, Extracranial, Childhood Germ Cell Tumor, ExtragonadalGerm Cell Tumor, Ovarian Gestational Trophoblastic Tumor Glioma, AdultGlioma, Childhood Brain Stem Glioma, Childhood Cerebral AstrocytomaGlioma, Childhood Visual Pathway and Hypothalamic Hairy Cell LeukemiaHead and Neck Cancer Hepatocellular (Liver) Cancer, Adult (Primary)Hepatocellular (Liver) Cancer, Childhood (Primary) Hodgkin's Lymphoma,Adult Hodgkin's Lymphoma, Childhood Hodgkin's Lymphoma During PregnancyHypopharyngeal Cancer Hypothalamic and Visual Pathway Glioma, ChildhoodIntraocular Melanoma Islet Cell Carcinoma (Endocrine Pancreas) Kaposi'sSarcoma Kidney (Renal Cell) Cancer Kidney Cancer, Childhood LaryngealCancer Laryngeal Cancer, Childhood Leukemia, Acute Lymphoblastic, AdultLeukemia, Acute Lymphoblastic, Childhood Leukemia, Acute Myeloid, AdultLeukemia, Acute Myeloid, Childhood Leukemia, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Hairy Cell Lip and Oral CavityCancer Liver Cancer, Adult (Primary) Liver Cancer, Childhood (Primary)Lung Cancer, Non-Small Cell Lung Cancer, Small Cell Lymphoma,AIDS-Related Lymphoma, Burkitt's Lymphoma, Cutaneous T-Cell, see MycosisFungoides and Sézary Syndrome Lymphoma, Hodgkin's, Adult Lymphoma,Hodgkin's, Childhood Lymphoma, Hodgkin's During Pregnancy Lymphoma,Non-Hodgkin's, Adult Lymphoma, Non-Hodgkin's, Childhood Lymphoma,Non-Hodgkin's During Pregnancy Lymphoma, Primary Central Nervous SystemMacroglobulinemia, Waldenström's Malignant Fibrous Histiocytoma ofBone/Osteosarcoma Medulloblastoma, Childhood Melanoma Melanoma,Intraocular (Eye) Merkel Cell Carcinoma Mesothelioma, Adult MalignantMesothelioma, Childhood Metastatic Squamous Neck Cancer with OccultPrimary Multiple Endocrine Neoplasia Syndrome, Childhood MultipleMyeloma/Plasma Cell Neoplasm Mycosis Fungoides Myelodysplastic SyndromesMyelodysplastic/Myeloproliferative Diseases Myelogenous Leukemia,Chronic Myeloid Leukemia, Adult Acute Myeloid Leukemia, Childhood AcuteMyeloma, Multiple Myeloproliferative Disorders, Chronic Nasal Cavity andParanasal Sinus Cancer Nasopharyngeal Cancer Nasopharyngeal Cancer,Childhood Neuroblastoma Non-Hodgkin's Lymphoma, Adult Non-Hodgkin'sLymphoma, Childhood Non-Hodgkin's Lymphoma During Pregnancy Non-SmallCell Lung Cancer Oral Cancer, Childhood Oral Cavity Cancer, Lip andOropharyngeal Cancer Osteosarcoma/Malignant Fibrous Histiocytoma of BoneOvarian Cancer, Childhood Ovarian Epithelial Cancer Ovarian Germ CellTumor Ovarian Low Malignant Potential Tumor Pancreatic Cancer PancreaticCancer, Childhood Pancreatic Cancer, Islet Cell Paranasal Sinus andNasal Cavity Cancer Parathyroid Cancer Penile Cancer PheochromocytomaPineoblastoma and Supratentorial Primitive Neuroectodermal Tumors,Childhood Pituitary Tumor Plasma Cell Neoplasm/Multiple MyelomaPleuropulmonary Blastoma Pregnancy and Breast Cancer Pregnancy andHodgkin's Lymphoma Pregnancy and Non-Hodgkin's Lymphoma Primary CentralNervous System Lymphoma Prostate Cancer Rectal Cancer Renal Cell(Kidney) Cancer Renal Cell (Kidney) Cancer, Childhood Renal Pelvis andUreter, Transitional Cell Cancer Retinoblastoma Rhabdomyosarcoma,Childhood Salivary Gland Cancer Salivary Gland Cancer, ChildhoodSarcoma, Ewing's Family of Tumors Sarcoma, Kaposi's Sarcoma, SoftTissue, Adult Sarcoma, Soft Tissue, Childhood Sarcoma, Uterine SezarySyndrome Skin Cancer (non-Melanoma) Skin Cancer, Childhood Skin Cancer(Melanoma) Skin Carcinoma, Merkel Cell Small Cell Lung Cancer SmallIntestine Cancer Soft Tissue Sarcoma, Adult Soft Tissue Sarcoma,Childhood Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma)Squamous Neck Cancer with Occult Primary, Metastatic Stomach (Gastric)Cancer Stomach (Gastric) Cancer, Childhood Supratentorial PrimitiveNeuroectodermal Tumors, Childhood T-Cell Lymphoma, Cutaneous, seeMycosis Fungoides and Sézary Syndrome Testicular Cancer Thymoma,Childhood Thymoma and Thymic Carcinoma Thyroid Cancer Thyroid Cancer,Childhood Transitional Cell Cancer of the Renal Pelvis and UreterTrophoblastic Tumor, Gestational Unknown Primary Site, Carcinoma of,Adult Unknown Primary Site, Cancer of, Childhood Unusual Cancers ofChildhood Ureter and Renal Pelvis, Transitional Cell Cancer UrethralCancer Uterine Cancer, Endometrial Uterine Sarcoma Vaginal Cancer VisualPathway and Hypothalamic Glioma, Childhood Vulvar Cancer Waldenström'sMacroglobulinemia Wilms' Tumor

In some embodiments, the proliferation disorder treated and/or monitoredusing the methods of the invention is prostate cancer. In someembodiments, the prostate cancer is a pre-cancer of the prostate. Insome embodiments, the prostate cancer is metastatic. In someembodiments, the prostate cancer is non-metastatic. In some embodiments,the prostate cancer is one that exhibits elevated expression of a HhPmember or ligand (i.e., a HhP-associated cancer). In some embodiments,the prostate cancer is castration-resistant. In some embodiments, theprostate cancer is non-castration resistant. In some embodiments, theprostate cancer is metastatic, castration-resistant prostate cancer. Insome embodiments, the prostate cancer is non-metastatic,castration-resistant prostate cancer.

In some embodiments, the proliferation disorder treated and/or monitoredusing the methods of the invention is skin cancer, such as melanoma, ora non-melanoma, such as basal cell carcinoma (BCC). Thus, in someembodiments, the proliferation disorder treated and/or monitored usingthe methods of the invention is BCC, which is a nonmelanocytic skincancer (i.e., an epithelial tumor) and is the most common form of skincancer. In some embodiments, the BCC is a type selected from amongnodular BCC, cystic BCC, cicatricial BCC, infiltrative BCC, micronodularBCC, superficial BCC, pigmented BCC, Jacobi ulcer, fibroepithelioma ofPinkus, polyoid basal-cell carcinoma, pore-like BCC, or aberrant BCC. Insome embodiments, the BCC is sporadic BCC. In some embodiments, the BCCis hereditary BCC. In some embodiments, the subject has a BCC tumorequal to or greater than 4 mm.

In some embodiments, the proliferation disorder is lung cancer (stage I,stage II, stage Ma, stage IIIb, or stage IV). In some embodiments, thelung cancer is a non-small cell lung cancer (NSCLC), such as squamouscell carcinoma, non-squamous cell carcinoma, large cell carcinoma, andadenocarcinoma. In some embodiments, the lung cancer is small cell lungcancer (SCLC). In some embodiments, the lung cancer is non-squamous celllung carcinoma. In some embodiments, the lung cancer is mesothelioma(e.g., malignant pleural mesothelioma). In some embodiments, the lungcancer is late-stage metastatic NSCLC.

Optionally, one or more tests are performed before and/or aftertreatment of the lung cancer, such as bone scan, chest x-ray, completeblood count (CDC), CT scan, liver function tests, magnetic resonanceimaging (MRI), positron emission tomography (PET), sputum test, andthoracentesis. Optionally, a biopsy may be obtained before and/or aftertreatment of the lung cancer (e.g., bronchoscopy with biopsy, CT-scandirected needle biopsy, endoscopic esophageal ultrasound with biopsy,mediastinoscopy with biopsy, open lung biopsy, pleural biopsy, and videoassisted thoracoscopy).

In some embodiments, the proliferation disorder to be treated isprostate cancer e.g., non-metastatic castrate resistant prostate canceror other prostate cancer. In some embodiments, the prostate cancer istreated by administering an HhP inhibitor such as itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, at a dose in the range of 100 mg to 600 mg per day.In some embodiments, the prostate cancer is treated by administering 200mg of an HhP inhibitor such as itraconazole, or a pharmaceuticallyacceptable salt, prodrug, stereoisomer, or active metabolite thereof,two or more times per day. Preferably, the HhP inhibitor such asitraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite thereof, is orally administered in aSUBA® formulation.

In some embodiments, the subject being treated for prostate cancer hasundergone androgen deprivation therapy, undergoes androgen deprivationtherapy concurrently with the HhP inhibitor treatment, or both. The goalof androgen deprivation therapy is to reduce androgen levels in the bodyor to prevent androgen from reaching prostate cancer cells. Examples oftreatments/agents for androgen deprivation therapy that may be utilizedinclude, but are not limited, to orchiectomy (surgical castration),luteinizing hormone-releasing hormone (LHRH) analogs (e.g., leuprolide,goserelin, triptorelin, or histrelin), luteinizing hormone-releasinghormone (LHRH) antagonists (e.g., degarelix and abiraterone),anti-androgens (flutamide, bicalutamide, nilutamide, and enzalutamide),and other androgen-suppressing drugs (e.g., ketoconazole).

In some embodiments, the proliferation disorder to be treated is basalcell carcinoma (BCC). In some embodiments, the BCC is treated byadministering an HhP inhibitor such as itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, at a dose in the range of 100 mg to 600 mg per day.In some embodiments, the BCC is treated by administering 150 mg of anHhP inhibitor such as itraconazole, or a pharmaceutically acceptablesalt, prodrug, stereoisomer, or active metabolite thereof, two or moretimes per day. Preferably, the HhP inhibitor such as itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, is orally administered in a SUBA® formulation. Insome embodiments, the subject being treated for BCC has a tumor equal toor greater than 4 mm.

In some embodiments, the proliferation disorder to be treated is lungcancer, e.g., late stage metastatic non-squamous non-small cell lungcancer or other lung cancer. In some embodiments, the lung cancer istreated by administering an HhP inhibitor such as itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, at a dose in the range of 100 mg to 600 mg per day.In some embodiments, the lung cancer is treated by administering 200 mgof an HhP inhibitor such as itraconazole, or a pharmaceuticallyacceptable salt, prodrug, stereoisomer, or active metabolite thereof,two or more times per day. Preferably, the HhP inhibitor such asitraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite thereof, is orally administered in aSUBA® formulation. Optionally, the method further comprisesadministration of an antifolate agent, such as pemetrexed, with orwithout a platinum-based agent, such as cisplatin as described inCombination Treatments. For example, without limitation, 300 mg/m²-700mg/m² of the antifolate agent and 25 mg/m²-125 mg/m² of theplatinum-based agent may be administered intravenously. In someembodiments, 500 mg/m² pemetrexed and 75 mg/m² cisplatin areadministered intravenously.

It has been demonstrated that HhP inhibitors (e.g., itraconazole) arecapable of delaying or inhibiting tumor cell growth. Using the methodsof the invention, the HhP inhibitors can be administered locally at thesite of a tumor (e.g., by direct injection) or remotely from the site(e.g., systemically). As used herein, the term “tumor” refers to allneoplastic cell growth and proliferation, whether malignant or benign,and all pre-cancerous and cancerous cells and tissues. For example, aparticular cancer may be characterized by a solid mass tumor ornon-solid tumor. The solid tumor mass, if present, may be a primarytumor mass. A primary tumor mass refers to a growth of cancer cells in atissue resulting from the transformation of a normal cell of thattissue. In most cases, the primary tumor mass is identified by thepresence of a cyst, which can be found through visual or palpationmethods, or by irregularity in shape, texture or weight of the tissue.However, some primary tumors are not palpable and can be detected onlythrough medical imaging techniques such as X-rays (e.g., mammography) ormagnetic resonance imaging (MM), or by needle aspirations. The use ofthese latter techniques is more common in early detection. Molecular andphenotypic analysis of cancer cells within a tissue can usually be usedto confirm if the cancer is endogenous to the tissue or if the lesion isdue to metastasis from another site.

Combination Treatments

According to the method of the subject invention, an HhP inhibitor canbe administered to a subject by itself, or co-administered with one ormore other agents such as an HhP inhibitor, or a different agent oragents. In some embodiments, the additional agent is one or moreanti-cancer agents. Anti-cancer agents include but are not limited tothe chemotherapeutic agents listed in Table 2.

Co-administration can be carried out simultaneously (in the same orseparate formulations) or consecutively with the additional agentadministered before and/or after one or more HhP inhibitors.Furthermore, HhP inhibitors can be administered to a subject as adjuvanttherapy. For example, one or more HhP inhibitors can be administered toa patient in conjunction with one or more chemotherapeutic agents.

Thus, the HhP inhibitor(s), whether administered separately, or as apharmaceutical composition, can include various other components asadditives. Examples of acceptable components or adjuncts which can beemployed in relevant circumstances include antioxidants, free radicalscavenging agents, peptides, growth factors, antibiotics, bacteriostaticagents, immunosuppressives, anticoagulants, buffering agents,anti-inflammatory agents, anti-angiogenics, anti-pyretics, time-releasebinders, anesthetics, steroids, and corticosteroids. Such components canprovide additional therapeutic benefit, act to affect the therapeuticaction of the HhP inhibitor, or act towards preventing any potentialside effects which may be posed as a result of administration of theseagents. The HhP inhibitor can be conjugated to a therapeutic agent, aswell.

In some embodiments, two or more HhP inhibitors are administered to thesubject simultaneously in the same or different formulations, orsequentially. The HhP inhibitors may act on the same member of the HhP,whether in similar or distinct manners, or on different members of thepathway. For example, it may be desirable to administer HhP inhibitorsthat inhibit the HhP pathway at different points in the pathway or bydifferent mechanisms. For example, while both itraconazole andvismodegib target Smo, they differ in the way they bind and act on thereceptor, inhibiting the HhP by different mechanisms of action.Vismodegib acts as a cylcopamine-competitive antagonist of the Smoreceptor, causing the transcription factors Gli1 and Gli2 to remaininactive, which inhibits the expression of tumor mediating genes withinthe HhP. In contrast, itraconazole inhibits activation of the HhP bytargeting Smo at a site distinct from that of cyclopamine mimicscurrently in development. The Smo protein can generally be activated byits translocation to the primary cilium and/or by changing itsconfiguration. Vismodegib works on Smo effectively by ensuring that theprotein does not change its configuration, whereas itraconazole works bypreventing its translocation. These distinctions are supported by theability of these two drugs to synergize. Accordingly, in someembodiments, one or more additional HhP inhibitors are administered andthe additional HhP inhibitor differs from the first HhP inhibitor in itsmechanism of action by which it inhibits the HhP (e.g., itraconazole, ora pharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite of itraconazole, and vismodegib, or a pharmaceuticallyacceptable salt, prodrug, stereoisomer, or active metabolite ofvismodegib).

Additional agents that can be co-administered to target cells in vitroor in vivo, such as in a subject, in the same or as a separateformulation, include those that modify a given biological response, suchas immunomodulators. The additional agents may be, for example, smallmolecules, polypeptides (proteins, peptides, or antibodies or antibodyfragments), or nucleic acids (encoding polypeptides or inhibitorynucleic acids such as antisense oligonucleotides or interfering RNA).For example, proteins such as tumor necrosis factor (TNF), interferon(such as alpha-interferon and beta-interferon), nerve growth factor(NGF), platelet derived growth factor (PDGF), and tissue plasminogenactivator can be administered. Biological response modifiers, such aslymphokines, interleukins (such as interleukin-1 (IL-1), interleukin-2(IL-2), and interleukin-6 (IL-6)), granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), or other growth factors can be administered. In one embodiment,the methods and compositions of the invention incorporate one or moreanti-cancer agents, such as cytotoxic agents, chemotherapeutic agents,anti-signaling agents, and anti-angiogenic agents.

As used herein, the term “anti-cancer agent” refers to a substance ortreatment (e.g., radiation therapy) that inhibits the function of cancercells, inhibits their formation, and/or causes their destruction invitro or in vivo. Examples include, but are not limited to, cytotoxicagents (e.g., 5-fluorouracil, TAXOL), chemotherapeutic agents, andanti-signaling agents (e.g., the PI3K inhibitor LY). In one embodiment,the anti-cancer agent administered before, during, or afteradministration of the HhP inhibitor is a different HhP inhibitor.Anti-cancer agents include but are not limited to the chemotherapeuticagents listed in Table 2.

As used herein, the term “cytotoxic agent” refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells in vitro and/or in vivo. The term is intended to includeradioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², and radioactive isotopes of Lu), chemotherapeutic agents,toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, and antibodies, includingfragments and/or variants thereof.

As used herein, the term “chemotherapeutic agent” is a chemical compounduseful in the treatment of cancer, such as, for example, taxanes, e.g.,paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil,vincristine, vinblastine, anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON, GTx, Memphis, Tenn.), and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc.Examples of chemotherapeutic agents that may be used in conjunction withthe Hhp inhibitors are listed in Table 2. In some embodiments, thechemotherapeutic agent is one or more anthracyclines. Anthracyclines area family of chemotherapy drugs that are also antibiotics. Theanthracyclines act to prevent cell division by disrupting the structureof the DNA and terminate its function by: (1) intercalating into thebase pairs in the DNA minor grooves; and (2) causing free radical damageof the ribose in the DNA. The anthracyclines are frequently used inleukemia therapy. Examples of anthracyclines include daunorubicin(CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX), epirubicin (ELLENCE,PHARMORUBICIN), and idarubicin (IDAMYCIN).

TABLE 2 Examples of Chemotherapeutic Agents   13-cis-Retinoic Acid2-Amino-6-Mercaptopurine 2-CdA 2-Chlorodeoxyadenosine 5-fluorouracil5-FU 6-TG 6-Thioguanine 6-Mercaptopurine 6-MP Accutane Actinomycin-DAdriamycin Adrucil Agrylin Ala-Cort Aldesleukin Alemtuzumab AlitretinoinAlkaban-AQ Alkeran All-transretinoic acid Alpha interferon AltretamineAmethopterin Amifostine Aminoglutethimide Anagrelide AnandronAnastrozole Arabinosylcytosine Ara-C Aranesp Aredia Arimidex AromasinArsenic trioxide Asparaginase ATRA Avastin BCG BCNU BevacizumabBexarotene Bicalutamide BiCNU Blenoxane Bleomycin Bortezomib BusulfanBusulfex C225 Calcium Leucovorin Campath Camptosar Camptothecin-11Capecitabine Carac Carboplatin Carmustine Carmustine wafer Casodex CCNUCDDP CeeNU Cerubidine cetuximab Chlorambucil Cisplatin Citrovorum FactorCladribine Cortisone Cosmegen CPT-11 Cyclophosphamide CytadrenCytarabine Cytarabine liposomal Cytosar-U Cytoxan DacarbazineDactinomycin Darbepoetin alfa Daunomycin Daunorubicin Daunorubicinhydrochloride Daunorubicin liposomal DaunoXome Decadron Delta-CortefDeltasone Denileukin diftitox DepoCyt Dexamethasone Dexamethasoneacetate dexamethasone sodium phosphate Dexasone Dexrazoxane DHAD DICDiodex Docetaxel Doxil Doxorubicin Doxorubicin liposomal Droxia DTICDTIC-Dome Duralone Efudex Eligard Ellence Eloxatin Elspar EmcytEpirubicin Epoetin alfa Erbitux Erwinia L-asparaginase EstramustineEthyol Etopophos Etoposide Etoposide phosphate Eulexin Evista ExemestaneFareston Faslodex Femara Filgrastim Floxuridine Fludara FludarabineFluoroplex Fluorouracil Fluorouracil (cream) Fluoxymesterone FlutamideFolinic Acid FUDR Fulvestrant G-CSF Gefitinib Gemcitabine Gemtuzumabozogamicin Gemzar Gleevec Lupron Lupron Depot Matulane MaxidexMechlorethamine Mechlorethamine Hydrochlorine Medralone Medrol MegaceMegestrol Megestrol Acetate Melphalan Mercaptopurine Mesna MesnexMethotrexate Methotrexate Sodium Methylprednisolone Mylocel LetrozoleNeosar Neulasta Neumega Neupogen Nilandron Nilutamide Nitrogen MustardNovaldex Novantrone Octreotide Octreotide acetate Oncospar Oncovin OntakOnxal Oprevelkin Orapred Orasone Oxaliplatin Paclitaxel PamidronatePanretin Paraplatin Pediapred PEG Interferon Pegaspargase PegfilgrastimPEG-INTRON PEG-L-asparaginase Phenylalanine Mustard Platinol Platinol-AQPrednisolone Prednisone Prelone Procarbazine PROCRIT ProleukinProlifeprospan 20 with Carmustine implant Purinethol RaloxifeneRheumatrex Rituxan Rituximab Roveron-A (interferon alfa-2a) -RubexRubidomycin hydrochloride Sandostatin Sandostatin LAR SargramostimSolu-Cortef Solu-Medrol STI-571 Streptozocin Tamoxifen Targretin TaxolTaxotere Temodar Temozolomide Teniposide TESPA Thalidomide ThalomidTheraCys Thioguanine Thioguanine Tabloid Thiophosphoamide ThioplexThiotepa TICE Toposar Topotecan Toremifene Trastuzumab Tretinoin TrexallTrisenox TSPA VCR Velban Velcade VePesid Vesanoid Viadur VinblastineVinblastine Sulfate Vincasar Pfs Vincristine Vinorelbine Vinorelbinetartrate VLB VP-16 Vumon Xeloda Zanosar Zevalin Zinecard ZoladexZoledronic acid Zometa Gliadel wafer Glivec GM-CSF Goserelingranulocyte—colony stimulating factor Granulocyte macrophage colonystimulating factor Halotestin Herceptin Hexadrol HexalenHexamethylmelamine HMM Hycamtin Hydrea Hydrocort Acetate HydrocortisoneHydrocortisone sodium phosphate Hydrocortisone sodium succinateHydrocortone phosphate Hydroxyurea Ibritumomab Ibritumomab TiuxetanIdamycin Idarubicin Ifex IFN-alpha Ifosfamide IL-2 IL-11 Imatinibmesylate Imidazole Carboxamide Interferon alfa Interferon Alfa-2b (PEGconjugate) Interleukin-2 Interleukin-11 Intron A (interferon alfa-2b)Leucovorin Leukeran Leukine Leuprolide Leurocristine Leustatin LiposomalAra-C Liquid Pred Lomustine L-PAM L-Sarcolysin Meticorten MitomycinMitomycin-C Mitoxantrone M-Prednisol MTC MTX Mustargen Mustine MutamycinMyleran Iressa Irinotecan Isotretinoin Kidrolase Lanacort L-asparaginaseLCR Pemetrexed

In some embodiments, an antifolate agent (e.g., a pyrimidine-basedantifolate agent), such as Pemetrexed, is administered to the subject,before, during, or after administration of the HhP inhibitor. Pemetrexedis a synthetic pyrimidine-based antifolate. Pemetrexed is also known asLY231514 and(2S)-2-{[4-[2-(2-amino-4-oxo-1,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino}pentanedioicacid, and is marked under the brand nameN-[4-2-(2-Amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-1-glutamicacid disodium salt (CAS Number: 150399-23-8). Pemetrexed binds to andinhibits the enzyme thymidylate synthase (TS), which catalyzes themethylation of 2′-deoxyduridine-5′-monophosphate (dUMP) to2′-deoxythymidine-5′-monophosphate (dTMP), an essential precursor in DNAsynthesis.

In some embodiments, a platinum-based agent (coordination complex ofplatinum) is administered to the subject before, during, or afteradministration of the HhP inhibitor. As a class, platinum-based agentsare believed to act by causing crosslinking of DNA as a monoadduct,interstrand crosslinks, intrastrand crosslinks, or DNA proteincrosslinks, resulting in inhibited DNA repair. In some embodiments, theplatinum-based agent is carboplatin, cisplatin, or oxaliplatin,satraplatin, picoplatin, nedaplatin, and triplatin.

Addition of an HhP inhibitor to a lung cancer treatment regimenincluding an antifolate such as pemetrexed can significantly increasethe subject's survival time (see Rudin et al., “Phase 2 Study ofPemetrexed and Itraconazole as Second-Line Therapy for MetastaticNonsquamous Non-Small-Cell Lung Cancer,” J. Thorac. Oncol., 2013,8(5):619-623, which is incorporated herein by reference in itsentirety). In some embodiments of the methods of the invention, theproliferation disorder to be treated is non-squamous NSCLC and thesubject is orally administered a SUBA® formulation of itraconazole(e.g., 100 mg to 600 mg per day of a SUBA® formulation), or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, two or more times per day. Optionally, the subjectis also administered an antifolate agent, such as pemetrexed, with orwithout a platinum-based agent, such as cisplatin by any appropriateroute. For example, without limitation, 300 mg/m²-700 mg/m² of theantifolate agent and 25 mg/m²-125 mg/m² of the platinum-based agent maybe administered intravenously. In some embodiments, 500 mg/m² pemetrexedand 75 mg/m² cisplatin are administered intravenously.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology that arewithin the skill of the art. Such techniques are explained fully in theliterature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to MolecularCloning (1984); the series, Methods In Enzymology (S. Colowick and N.Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hameset al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H.Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification: Principles and Practice(2nd ed., Springer-Verlag); and PCR: A Practical Approach (McPherson etal. Eds. (1991) IRL Press)), each of which are incorporated herein byreference in their entirety.

Experimental controls are considered fundamental in experiments designedin accordance with the scientific method. It is routine in the art touse experimental controls in scientific experiments to prevent factorsother than those being studied from affecting the outcome.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

EXEMPLIFIED EMBODIMENTS Embodiment 1

A method for treating a proliferation disorder in a subject, comprisingadministering a composition comprising a Hedgehog pathway (HhP)inhibitor to the subject, wherein the composition is administered in aneffective amount to achieve a plasma trough level of at least about1,000 ng/mL of the HhP inhibitor.

Embodiment 2

The method of embodiment 1, wherein the HhP inhibitor comprisesitraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite thereof.

Embodiment 3

The method of embodiment 2, wherein the composition comprises a SUBA®formulation of itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof; and wherein theSUBA® formulation is orally administered at a dose in the range of 100mg to 600 mg itraconazole per day.

Embodiment 4

The method of embodiment 2, wherein the HhP inhibitor therapy comprisesadministration of a capsule or powder of 50 mg of the itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, twice per day.

Embodiment 5

The method of embodiment 2, wherein the SUBA® formulation is a SUBA-CAP™formulation.

Embodiment 6

The method of any preceding embodiment, wherein the composition isadministered in an effective amount to achieve a plasma trough level ofat least 1,000 ng/mL of the HhP inhibitor.

Embodiment 7

The method of any preceding embodiment, wherein the composition isadministered in an effective amount to achieve a plasma trough level ofat least about 1,000 ng/mL of the HhP inhibitor after about 4 weeks ofinitiation of treatment with the HhP inhibitor.

Embodiment 8

The method of any preceding embodiment, wherein the composition isadministered in an effective amount to achieve a plasma trough level ofat least about 1,000 ng/mL of the HhP inhibitor within about 2 weeksafter initiation of treatment, and to maintain the plasma trough levelof at least about 1,000 ng/mL of the HhP inhibitor for the duration ofthe treatment.

Embodiment 9

The method of any preceding embodiment, further comprising measuring theplasma level of the HhP inhibitor, or a metabolite thereof, in thesubject one or more times.

Embodiment 10

The method of embodiment 9, wherein said measuring is carried out one ormore times about 4 weeks after initiation of treatment with the HhPinhibitor.

Embodiment 11

The method of any preceding embodiment, further comprising measuring theplasma level of the HhP inhibitor, or a metabolite thereof, one or moretimes in a period of time from about 4 weeks to about 12 weeks.

Embodiment 12

The method of embodiment 11, further comprising increasing a subsequentdose of the HhP inhibitor if the plasma trough level of at least about1,000 ng/mL of the HhP inhibitor is not maintained.

Embodiment 13

The method of embodiment 11, further comprising reducing a subsequentdose of an HhP inhibitor if the plasma trough level at about 4 weeks isat least 1000 ng/mL and the patient is experiencing one or more sideeffects.

Embodiment 14

The method of any preceding embodiment, wherein the HhP inhibitor isadministered at least once daily.

Embodiment 15

The method of any preceding embodiment, wherein the HhP inhibitor isadministered at least twice daily.

Embodiment 16

The method of any preceding embodiment, wherein the proliferationdisorder is cancer.

Embodiment 17

The method of embodiment 16, wherein the cancer is lung cancer.

Embodiment 18

The method of embodiment 16, wherein the cancer is basal cell carcinoma(BCC).

Embodiment 19

The method of embodiment 16, wherein the cancer is prostate cancer.

Embodiment 20

The method of embodiment 16, wherein the cancer is prostate cancer andsaid method further comprises comparing the level of prostate-specificantigen (PSA) in a sample obtained from the subject followingadministration of the HhP inhibitor with a reference level of PSA, andwherein the level of PSA in the sample compared to the reference levelof PSA is prognostic for an outcome of treatment with the HhP inhibitor.

Embodiment 21

The method of embodiment 20, wherein a PSA level increase of less thanabout 25% relative to the PSA level at initiation of HhP inhibitortreatment is indicative of efficacy and a PSA level increase of about25% or greater is indicative of a lack of efficacy.

Embodiment 22

The method of embodiment 20 or 21, wherein the sample is obtained fromthe subject within 4 to 12 weeks after initiation of HhP inhibitortherapy.

Embodiment 23

The method of embodiment 20 or 21, wherein the method further comprisesobtaining the sample from the subject after said administering.

Embodiment 24

The method of embodiment 20 or 21, wherein the method further comprisesmaintaining HhP inhibitor therapy if the measured level of PSA isindicative of efficacy.

Embodiment 25

The method of embodiment 20 or 21, wherein the method further comprisesceasing treatment with the HhP inhibitor if the measured level of PSA isindicative of a lack of efficacy.

Embodiment 26

The method of embodiment 25, further comprising administering atreatment for the prostate cancer other than an HhP inhibitor.

Embodiment 27

The method of embodiment 25, wherein the treatment comprises one or morefrom among radiation therapy, hormone therapy, chemotherapy,immunotherapy, surgery, cryosurgery, high-intensity focused ultrasound,and proton beam radiation therapy.

Embodiment 28

The method of embodiment 20 or 21, wherein the method further comprisesincreasing the dose of the HhP inhibitor and/or frequency of dose of theHhP inhibitor if the measured level of PSA is indicative of a lack ofefficacy.

Embodiment 29

The method of embodiment 20 or 21, wherein the method further comprisesdecreasing the dose of the HhP inhibitor and/or frequency of dose of theHhP inhibitor if the measured level of PSA is indicative of efficacy butthe subject is experiencing one or more adverse effects.

Embodiment 30

The method of embodiment 20 or 21, wherein the PSA level is the level oftotal PSA (free (unbound) PSA and bound PSA).

Embodiment 31

The method of embodiment 20 or 21, wherein the PSA level is PSA doublingtime.

Embodiment 32

The method of embodiment 20 or 21, wherein PSA protein level ismeasured.

Embodiment 33

The method of embodiment 32, wherein the PSA protein level is measuredby radioimmunoassay (MA), immunoradiometric assay (IRMA), enzyme-linkedimmunosorbent assay (ELISA), dot blot, slot blot, enzyme-linkedimmunosorbent spot (ELISPOT) assay, Western blot, peptide microarray,surface plasmon resonance, fluorescence resonance energy transfer,bioluminescence resonance energy transfer, fluorescence quenchingfluorescence, fluorescence polarization, mass spectrometry (MS),high-performance liquid chromatography (HPLC), high-performance liquidchromatography/mass spectrometry (HPLC/MS), high-performance liquidchromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS),capillary electrophoresis, rod-gel electrophoresis, or slab-gelelectrophoresis.

Embodiment 34

The method of embodiment 20 or 21, wherein PSA DNA or mRNA level ismeasured.

Embodiment 35

The method of embodiment 34, wherein the PSA mRNA level is measured byNorthern blot, Southern blot, nucleic acid microarray, polymerase chainreaction (PCR), real time-PCR (RT-PCR), nucleic acid sequence basedamplification assay (NASBA), or transcription mediated amplification(TMA).

Embodiment 36

The method of embodiment 20 or 21, wherein PSA activity level ismeasured.

Embodiment 37

The method of embodiment 20 or 21, wherein the sample is a serum sample.

Embodiment 38

The method of any one of embodiments 20 to 37, wherein the methodfurther comprises obtaining the sample from the subject.

Embodiment 39

The method of any preceding embodiment, further comprising monitoringthe proliferation disorder in the subject, wherein a lack of clinicalresponse in the proliferation disorder to the treatment is indicativethat the plasma trough level of the HhP inhibitor should be increasedfurther above about 1000 ng/mL, and wherein the occurrence of a clinicalresponse and a plasma trough level of the HhP inhibitor substantiallyhigher than about 1000 ng/mL indicates that one or more subsequent dosesof the HhP inhibitor can be reduced.

Embodiment 40

The method of any preceding embodiment, further comprising monitoringthe proliferation disorder in the subject, wherein a lack of clinicalresponse in the proliferation disorder to the treatment, after aboutfour weeks of said administering, is indicative of a need to increasethe dose, and/or frequency of the dose, of the HhP inhibitor.

Embodiment 41

The method of any preceding embodiment, further comprising monitoringthe proliferation disorder in the subject, wherein the occurrence of aclinical response in the proliferation disorder to the treatment, afterabout four weeks of said administering, is indicative of a need todecrease the dose, and/or frequency of the dose, of the HhP inhibitor.

Embodiment 42

The method of any one of embodiments 39-41, wherein said monitoringcomprises visual inspection, palpation, imaging, assaying the presence,level, or activity of one or more biomarkers associated with theproliferation disorder in a sample obtained from the subject, or acombination of two or more of the foregoing.

Embodiment 43

The method of embodiment 42, wherein the one or more biomarkers compriseGli1, Gli2, Gli3, or a combination of two or more of the foregoing.

Embodiment 44

The method of any one of embodiments 39-43, wherein said monitoringcomprises monitoring at least one of the following parameters: tumorsize, rate of change in tumor size, hedgehog levels or signaling,appearance of a new tumor, rate of appearance of new tumors, change in asymptom of the proliferation disorder, appearance of a new symptomassociated with the proliferation disorder, quality of life (e.g.,amount of pain associated with the proliferation disorder), or acombination of two or more of the foregoing.

Embodiment 45

The method of embodiment 20 or 21, wherein the method further comprisesmonitoring the PSA level in the subject, comprising comparing the PSAlevel in multiple samples with the reference level of PSA, wherein thesamples are obtained from the subject over time, following HhP inhibitortreatment.

Embodiment 46

The method of any preceding embodiment, further comprising administeringeplerenone or other mineralocorticoid inhibitor, or pemetrexed or otherantifolate, or cisplatin or other platinum-based chemotherapeutic agent,or a combination of two or more of the foregoing, to the subject.

Embodiment 47

The method of embodiment 46, wherein the subject is suffering from anadverse effect selected from hypertension, peripheral edema, andhypokalemia, and wherein the mineralocorticoid inhibitor is administeredin an amount effective to treat the adverse effect.

Embodiment 48

The method of any preceding embodiment, wherein the HhP inhibitor is apurified stereoisomer of itraconazole (non-racemic mixture), or anitraconazole analogue in which the sec-butyl side chain has beenreplaced with one or more moieties, relative to itraconazole.

Embodiment 49

The method of any preceding embodiment, wherein the subject has a fungalinfection.

Embodiment 50

The method of any one of embodiments 1 to 48, wherein the subject doesnot have a fungal invention.

Embodiment 51

The method of any one of embodiments 1 to 49, wherein the subject has afungal infection selected from Blastomycosis, Histoplasmosis,Candidiasis, and Aspergillosis.

Embodiment 52

The method of any one of embodiments 1 to 48, wherein the subject doesnot have a fungal infection selected from among Blastomycosis,Histoplasmosis, Candidiasis, and Aspergillosis.

Embodiment 53

The method of any preceding embodiment, wherein the subject has receivedno prior chemotherapy to treat the proliferation disorder.

Embodiment 54

The method of any preceding embodiment, wherein the subject isadministered no steroid during the duration of the treatment.

Embodiment 55

The method of any preceding embodiment, wherein the subject isadministered no agent that interacts with CYP3A4 during the duration ofthe treatment.

Embodiment 56

The method of any preceding embodiment, wherein the duration oftreatment with the HhP inhibitor is in the range of about 4 weeks toabout 24 weeks.

Embodiment 57

The method of any preceding embodiment, wherein the HhP inhibitortargets the Smoothened protein of the HhP pathway.

Embodiment 58

The method of any preceding embodiment, wherein the HhP inhibitor iscyclopamine-competitive.

Embodiment 59

The method of any preceding embodiment wherein the HhP inhibitor iscyclopamine-competitive and the proliferation disorder comprisesprostate cancer.

Embodiment 60

The method of embodiment 59, wherein the proliferation disorder isprostate cancer and said method further comprises comparing the level ofprostate-specific antigen (PSA) in a sample obtained from the subjectfollowing administration of the HhP inhibitor with a reference level ofPSA, and wherein the level of PSA in the sample compared to thereference level of PSA is prognostic for an outcome of treatment withthe HhP inhibitor.

Embodiment 61

The method of embodiment 60, wherein a PSA level increase of less thanabout 25% relative to the PSA level at initiation of HhP inhibitortreatment is indicative of efficacy and a PSA level increase of about25% or greater is indicative of a lack of efficacy.

Embodiment 62

A method of prognosticating an outcome of prostate cancer treatment witha Hedgehog pathway (HhP) inhibitor therapy in a subject, comprisingcomparing the level of prostate-specific antigen (PSA) in a sampleobtained from the subject following HhP inhibitor therapy with areference level of PSA, wherein the level of PSA in the sample comparedto the reference level of PSA is prognostic for an outcome of treatmentwith the HhP inhibitor.

Embodiment 63

The method of embodiment 62, wherein the reference level is the PSAlevel in the subject at initiation of HhP inhibitor therapy.

Embodiment 64

A method of determining the efficacy of Hedgehog pathway (HhP) inhibitortherapy for prostate cancer in a human subject, comprising measuringprostate-specific antigen (PSA) level in a sample obtained from thesubject following initiation of HhP inhibitor therapy, wherein ameasured PSA level compared to a first reference PSA level at initiationof HhP inhibitor therapy is indicative of efficacy, and wherein ameasured PSA level compared to a second reference PSA level isindicative of a lack of efficacy.

Embodiment 65

The method of embodiment 62 or 64, wherein a PSA level increase of lessthan about 25% relative to the PSA level at initiation of HhP inhibitortherapy is indicative of efficacy and a PSA level increase of about 25%or greater is indicative of a lack of efficacy.

Embodiment 66

The method of embodiment 62 or 64, wherein the HhP inhibitor is orallyadministered in an effective amount to achieve a plasma trough level ofat least about 1,000 ng/mL of the HhP inhibitor.

Embodiment 67

The method of embodiment 62 or 64, wherein the HhP inhibitor is orallyadministered in an effective amount to achieve a plasma trough level ofat least 1,000 ng/mL of the HhP inhibitor.

Embodiment 68

The method of embodiment 62 or 64, wherein the HhP inhibitor therapycomprises oral administration of the HhP inhibitor at a dose in therange of 100 mg to 600 mg per day.

Embodiment 69

The method of embodiment 62 or 64, wherein the HhP inhibitor targets theSmoothened protein of the HhP pathway.

Embodiment 70

The method of embodiment 62 or 64, wherein the HhP inhibitor iscyclopamine-competitive.

Embodiment 71

The method of embodiment 62 or 64, wherein the HhP inhibitor comprisesitraconazole, or a pharmaceutically acceptable salt, prodrug,stereoisomer, or active metabolite thereof.

Embodiment 72

The method of embodiment 62 or 64, wherein the HhP inhibitor comprises aSUBA® formulation (e.g., a SUBACAP™ formulation) of itraconazole.

Embodiment 73

The method of embodiment 62 or 64, wherein the HhP inhibitor comprises aSUBA® formulation of itraconazole, and wherein the HhP inhibitor therapycomprises administration of the SUBA® formulation at a dose oritraconazole in the range of 100 mg to 600 mg per day.

Embodiment 74

The method of any one of embodiments 71-73, wherein the HhP inhibitortherapy comprises administration of a capsule or powder of 50 mg of theHhP inhibitor twice per day.

Embodiment 75

The method of any preceding embodiment, wherein the HhP inhibitortherapy comprises administration of the HhP inhibitor intravenously orlocally (e.g., by direct injection) at the site of a prostate cancerlesion or tumor.

Embodiment 76

The method of embodiment 62 or 64, wherein the sample is obtained fromthe subject within 4 to 12 weeks after initiation of HhP inhibitortherapy.

Embodiment 77

The method of embodiment 62 or 64, wherein the method further comprisesadministering the HhP inhibitor to the subject, and obtaining the samplefrom the subject after said administering.

Embodiment 78

The method of embodiment 62 or 64, wherein the method further comprisesmaintaining HhP inhibitor therapy if the measured level of PSA isindicative of efficacy.

Embodiment 79

The method of embodiment 62 or 64, wherein the method further compriseswithholding HhP inhibitor therapy if the measured level of PSA isindicative of a lack of efficacy.

Embodiment 80

The method of embodiment 79, further comprising administering atreatment for the prostate cancer other than an HhP inhibitor.

Embodiment 81

The method of embodiment 80, wherein the treatment comprises one or morefrom among radiation therapy, hormone therapy, chemotherapy,immunotherapy, surgery, cryosurgery, high-intensity focused ultrasound,and proton beam radiation therapy.

Embodiment 82

The method of embodiment 62 or 64, wherein the method further comprisesincreasing the dose of the HhP inhibitor and/or frequency of dose of theHhP inhibitor if the measured level of PSA is indicative of a lack ofefficacy.

Embodiment 83

The method of embodiment 62 or 64, wherein the method further comprisesdecreasing the dose of the HhP inhibitor and/or frequency of dose of theHhP inhibitor if the measured level of PSA is indicative of efficacy butthe subject is experiencing one or more side effects

Embodiment 84

The method of embodiment 62 or 64, wherein the PSA level is the level oftotal PSA (free (unbound) PSA and bound PSA).

Embodiment 85

The method of embodiment 62 or 64, wherein the PSA level is PSA doublingtime.

Embodiment 86

The method of embodiment 62 or 64, wherein PSA protein level ismeasured.

Embodiment 87

The method of embodiment 86, wherein the PSA protein level is measuredby radioimmunoassay (MA), immunoradiometric assay (IRMA), enzyme-linkedimmunosorbent assay (ELISA), dot blot, slot blot, enzyme-linkedimmunosorbent spot (ELISPOT) assay, Western blot, peptide microarray,surface plasmon resonance, fluorescence resonance energy transfer,bioluminescence resonance energy transfer, fluorescence quenchingfluorescence, fluorescence polarization, mass spectrometry (MS),high-performance liquid chromatography (HPLC), high-performance liquidchromatography/mass spectrometry (HPLC/MS), high-performance liquidchromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS),capillary electrophoresis, rod-gel electrophoresis, or slab-gelelectrophoresis.

Embodiment 88

The method of embodiment 62 or 64, wherein PSA DNA or mRNA level ismeasured.

Embodiment 89

The method of embodiment 88, wherein the PSA mRNA level is measured byNorthern blot, Southern blot, nucleic acid microarray, polymerase chainreaction (PCR), real time-PCR (RT-PCR), nucleic acid sequence basedamplification assay (NASBA), or transcription mediated amplification(TMA).

Embodiment 90

The method of embodiment 62 or 64, wherein PSA activity level ismeasured.

Embodiment 91

The method of embodiment 62 or 64, wherein the sample is a serum sample.

Embodiment 92

The method of embodiment 62 or 64, wherein the method further comprisesobtaining the sample from the subject.

Embodiment 93

The method of embodiment 62, wherein the method comprises monitoring thePSA level in the subject, comprising comparing the PSA level in multiplesamples with the reference level of PSA, wherein the samples areobtained from the subject over time, following HhP inhibitor therapy.

Embodiment 94

The method of embodiment 64, wherein the method comprises monitoring thePSA level in the subject, comprising measuring the PSA level in multiplesamples obtained from the subject over time, following HhP inhibitortherapy.

Embodiment 95

A method for treating prostate cancer in a subject, comprisingadministering Hedgehog pathway (HhP) inhibitor therapy to the subject;and carrying out the method of any one of embodiments 62 to 94.

Embodiment 96

A method for determining a dose of HhP inhibitor suitable foradministration to a subject for treatment of prostate cancer, comprisingmeasuring a PSA level in a sample obtained from the subject; anddetermining an effective dose of HhP inhibitor based on comparison ofthe measured PSA level to a reference level of PSA.

Embodiment 97

The method of embodiment 95 or 96, wherein the HhP inhibitor iscyclopamine-competitive.

Embodiment 98

The method of any preceding embodiment, wherein the proliferationdisorder is prostate cancer, wherein the composition comprises a SUBA®formulation of itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, and wherein theSUBA® formulation is orally administered at a dose in the range of 100mg to 600 mg per day.

Embodiment 99

The method of embodiment 18, wherein the composition comprises a SUBA®formulation of itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, and wherein theSUBA® formulation is orally administered at a dose in the range of 100mg to 600 mg per day.

Embodiment 100

The method of embodiment 17, wherein the composition comprises a SUBA®formulation of itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, wherein the SUBA®formulation is orally administered at a dose in the range of 100 mg to600 mg per day, and wherein the method further comprises administeringan antifolate and a platinum-based chemotherapeutic agent to thesubject.

Embodiment 101

The method of embodiment 1, further comprising monitoring theproliferation disorder in the subject, wherein a lack of clinicalresponse in the proliferation disorder to the treatment, after aboutfour weeks of said administering, is indicative of a need to increasethe dose of the HhP inhibitor, or increase the frequency of the dose ofthe HhP inhibitor, or administer an additional HhP inhibitor thatinhibits the HhP by a different mechanism than the previouslyadministered HhP inhibitor, or a combination of two or more of theforegoing.

Embodiment 102

The method of embodiment 1, 62, 64, 95, or 96, wherein the HhP inhibitoris cyclopamine-competitive.

Embodiment 103

The method of embodiment 1, 62, 64, 95, or 96, wherein the HhP inhibitoris non-cyclopamine-competitive.

Embodiment 104

The method of embodiment 1, further comprising, prior to saidadministering, selecting the subject for treatment, wherein saidselecting comprises:

measuring a biomarker in a sample obtained from the subject, wherein thebiomarker is directly or indirectly representative of HhP signaling, and

comparing the measured biomarker level to a reference level to determinethe presence or absence of elevated HhP signaling, wherein the presenceof elevated HhP signaling indicates that the subject should be treated.

Embodiment 105

The method of embodiment 104, wherein the biomarker is an HhP ligand ora glioma-associated oncogene homolog (Gli) transcription factor (e.g.,Sonic hedgehog (SHH), desert hedgehog (DHH), Indian hedgehog (DHH),Gli1, Gli2, Gli3, or a combination or two or more of the foregoing).

Embodiment 106

The method of embodiment 1, further comprising monitoring theproliferation disorder in the subject, wherein said monitoringcomprises:

measuring a biomarker in a sample obtained from the subject after saidadministering, wherein the biomarker is directly or indirectlyrepresentative of HhP signaling, and

comparing the measured biomarker level to a reference level to determinewhether HhP signaling has increased, decreased, or has not changed aftersaid comparing, wherein an increase or no change in HhP signalingindicates a need to modify the treatment by increasing the dose of theHhP inhibitor, or increasing the frequency of the dosing of the HhPinhibitor, or administering an additional HhP inhibitor before, duringor after the currently administered HhP inhibitor, or a combination oftwo or more of the foregoing; and wherein a decrease in HhP signalingindicates that the HhP inhibitor dose, frequency of HhP inhibitor dose,and choice of HhP inhibitor currently being administered aresatisfactory and may proceed.

Embodiment 107

The method of embodiment 106, wherein the additional HhP inhibitor andHhP inhibitor currently being administered inhibit the HhP by differentmechanisms of action.

Embodiment 108

The method of embodiment 107, wherein the biomarker is an HhP ligand ora glioma-associated oncogene homolog (Gli) transcription factor (e.g.,Sonic hedgehog (SHH), desert hedgehog (DHH), Indian hedgehog (DHH),Gli1, Gli2, Gli3, or a combination of two or more of the foregoing).

Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the term “plasma trough level” refers to theconcentration of an agent (e.g., a HhP inhibitor) in plasma immediatelybefore the next dose, or the minimum concentration of the agent betweentwo doses.

As used herein, the terms “proliferation disorder”, “cell proliferationdisorder”, “proliferative disorder”, “cell proliferative disorder”,“condition characterized by undesirable cell proliferation”, andgrammatical variations thereof refer to any pathological ornon-pathological physiological condition characterized by aberrant orundesirable proliferation of at least one cell, including but notlimited to conditions characterized by undesirable or unwanted oraberrant cell proliferation, conditions characterized by undesirable orunwanted or aberrant cell survival, and conditions characterized bydeficient or aberrant apoptosis. The term “cell proliferation” andgrammatical variations thereof, is understood to encompass both anincrease in the number of cells as a result of cell division, as well asan increase in the total mass of cells as a result of cell growth, e.g.,by growth of daughter cells after mitosis. An example of a proliferationdisorder is cancer, e.g., undesirable or unwanted or aberrantproliferation and survival of cancer cells such as cells associated withprostate cancer, lymphoma, myeloma, sarcoma, leukemia, or otherneoplastic disorders disclosed elsewhere herein and known to one ofskill in the art. Proliferation disorders include pre-cancerous orpre-malignant conditions (e.g., morphologically identifiable lesionsthat precede invasive cancers), intraepithelial neoplasia (e.g.,prostatic IEN and cervical IEN), atypical adenomatous hyperplasia,colorectal polyps, basal cell nevus syndrome, actinic keratosis,Barrett's esophagus, atrophic gastritis, and cervical dysplasia.Examples of non-cancerous proliferation disorders include smooth musclecell proliferation, systemic sclerosis, cirrhosis of the liver, adultrespiratory distress syndrome, idiopathic cardiomyopathy, lupuserythematosus, retinopathy, (e.g., diabetic retinopathy or otherretinopathies), cardiac hyperplasia, reproductive system associateddisorders such as benign prostatic hyperplasia and ovarian cysts,pulmonary fibrosis, endometriosis, fibromatosis, hamartomas,lymphangiomatosis, sarcoidosis and desmoid tumors. Non-cancerousproliferation disorders also include hyperproliferation of cells in theskin such as psoriasis and its varied clinical forms, Reiter's syndrome,pityriasis rubra pilaris, hyper-proliferative variants of disorders ofkeratinization (e.g., actinic keratosis, senile keratosis), scleroderma,seborrheic keratoses, intraepidermal nevi, common warts, benignepithelial tumors, and the like.

The terms “cancer” and “malignancy” are used herein interchangeably torefer to or describe the physiological condition in mammals that istypically characterized by unregulated cell growth. The term encompassesdysplasia, carcinoma in situ (CIS), and carcinoma. The cancer may bemetastatic or non-metastatic.

As used herein, the term “prostate cancer” refers to cancer orpre-cancer of the prostate, including adenocarcinoma and small cellcarcinoma. The term encompasses prostatic intraepithelial neoplasia(PIN) and carcinoma in situ of the prostate. Typically, the prostatecancer will be one that exhibits elevated expression of a Hedgehogpathway member or ligand (i.e., a Hedgehog pathway-associated cancer).The prostate cancer may be metastatic or non-metastatic. The prostatecancer may be castration-resistant or non-castration resistant. In someembodiments, the prostate cancer is metastatic, castration-resistantprostate cancer. In some embodiments, the prostate cancer isnon-metastatic, castration-resistant prostate cancer.

As used herein, the term “Gli” refers to any one of the Gli1, Gli2 orGli3 proteins, or a combination of two or more of the foregoing. “gli”refers to the nucleic acid encoding the Gli proteins, and gli1, gli2 andgli3 are the genes encoding the Gli1, Gli2 and Gli3 proteins.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.For example, “an HhP inhibitor” encompasses one or more HhP inhibitors,“a sample” encompasses one or more samples, etc.

As used herein, the term “or” is used herein to mean, and is usedinterchangeably with, the term “and/or”, unless context clearlyindicates otherwise.

As used herein, the terms “about” and “approximately” shall generallymean an acceptable degree of error for the quantity measured given thenature or precision of the measurements. Exemplary degrees of error arewithin 20 percent (%), typically, within 10%, and more typically, within5% of a given value or range of values.

As used herein, the terms “patient”, “subject”, and “individual” areused interchangeably and are intended to include males of the human andnon-human animal species. For example, the subject may be a human or ananimal model.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject has cancer (as therapy), or before the subject has cancer (asprophylaxis), which reduces the severity of the cancer, retards or slowsthe progression of the cancer, or prevents the cancer. Thus treatmentwith HhP inhibitors may prevent or manage cancer.

As used herein, unless otherwise specified, the terms “prevent,”“preventing”, and “prevention” contemplate an action that occurs beforea subject begins to suffer from the regrowth of the cancer and/or whichinhibits or reduces the severity of the cancer, or delays its onset.

As used herein, and unless otherwise specified, the terms “manage,”“managing” and “management” encompass preventing the recurrence of thecancer in a subject who has already suffered from the cancer, and/orlengthening the time that a subject who has suffered from the cancerremains in remission. The terms encompass modulating the threshold,development and/or duration of the cancer, or changing the way that apatient responds to the cancer.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound (e.g., an HhP inhibitor) is an amountsufficient to provide a therapeutic benefit in the treatment ormanagement of the proliferation disorder (e.g., cancer), or to delay orminimize one or more symptoms associated with the proliferationdisorder. A therapeutically effective amount of a compound means anamount of therapeutic agent, alone or in combination with othertherapeutic agents, which provides a therapeutic benefit in thetreatment or management of the proliferation disorder. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of theproliferation disorder, or enhances the therapeutic efficacy of anothertherapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound (e.g., a HhP inhibitor) is an amountsufficient to prevent regrowth of the proliferation disorder (e.g.,cancer), or one or more symptoms associated with the proliferationdisorder, or prevent its recurrence. A prophylactically effective amountof a compound means an amount of the compound, alone or in combinationwith other therapeutic agents, which provides a prophylactic benefit inthe prevention of the proliferation disorder. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

As used herein, the term “efficacy” in the context of HhP inhibitorytherapy refers to the ability of the therapy (as monotherapy or incombination therapy with another HhP inhibitor or other agent that isnot an HhP inhibitor) to alleviate one or more symptoms of theproliferation disorder (e.g., cancer), diminish the extent of disease,stabilize (i.e., not worsening) the state of the disease, delay or slowdisease progression, amelioration or palliation of the disease state,remission (whether partial or total), whether detectable orundetectable, tumor regression, inhibit tumor growth, inhibit tumormetastasis, reduce cancer cell number, inhibit cancer cell infiltrationinto peripheral organs, increase progression free survival, improveprogression free survival, improve time to disease progression (TTP),improve response rate (RR), prolonged overall survival (OS), prolongtime-to-next-treatment (TNTT), or prolong time from first progression tonext treatment, or a combination of two or more of the foregoing.

As used herein, the terms “anticancer agent,” “conventional anticanceragent,” or “cancer therapeutic drug” refer to any therapeutic agents(e.g., chemotherapeutic compounds and/or molecular therapeuticcompounds), radiation therapies, or surgical interventions, used in thetreatment of cancer (e.g., in mammals). HhP inhibitors may beadministered with a therapeutic agent, such as an anticancer agent.

As used herein, the terms “drug” and “chemotherapeutic agent” refer topharmacologically active molecules that are used to diagnose, treat, orprevent diseases or pathological conditions in a physiological system(e.g., a subject, or in vivo, in vitro, or ex vivo cells, tissues, andorgans). Drugs act by altering the physiology of a living organism,tissue, cell, or in vitro system to which the drug has beenadministered. It is intended that the terms “drug” and “chemotherapeuticagent” encompass anti-hyperproliferative and antineoplastic compounds aswell as other biologically therapeutic compounds.

As used herein, the term “solvate” refers to an HhP inhibitor havingeither a stoichiometric or non-stoichiometric amount of a solventassociated with the compound. The solvent can be water (i.e., ahydrate), and each molecule of inhibitor can be associated with one ormore molecules of water (e.g., monohydrate, dihydrate, trihydrate,etc.). The solvent can also be an alcohol (e.g., methanol, ethanol,propanol, isopropanol, etc.), a glycol (e.g., propylene glycol), anether (e.g., diethyl ether), an ester (e.g., ethyl acetate), or anyother suitable solvent. The hedgehog inhibitor can also exist as a mixedsolvate (i.e., associated with two or more different solvents).

We claim:
 1. A method for treating a proliferation disorder in asubject, comprising orally administering a composition comprising aHedgehog pathway (HhP) inhibitor to the subject, wherein the compositionis orally administered in an effective amount to achieve a plasma troughlevel of at least about 1,000 ng/mL of the HhP inhibitor.
 2. The methodof claim 1, wherein the HhP inhibitor comprises itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof.
 3. The method of claim 2, wherein the compositioncomprises a SUBA® formulation of itraconazole, or a pharmaceuticallyacceptable salt, prodrug, stereoisomer, or active metabolite thereof andwherein the SUBA® formulation is orally administered at a dose in therange of 100 mg to 600 mg per day.
 4. The method of claim 2, wherein theHhP inhibitor therapy comprises administration of a capsule or powder of50 mg of the itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, twice per day. 5.The method of claim 3, wherein the SUBA® formulation is a SUBA-CAPformulation.
 6. The method of claim 1, wherein the composition isadministered in an effective amount to achieve a plasma trough level ofat least 1,000 ng/mL of the HhP inhibitor.
 7. The method of claim 1,wherein the composition is administered in an effective amount toachieve a plasma trough level of at least about 1,000 ng/mL of the HhPinhibitor after about 4 weeks of initiation of treatment with the HhPinhibitor.
 8. The method of claim 1, wherein the composition isadministered in an effective amount to achieve a plasma trough level ofat least about 1,000 ng/mL of the HhP inhibitor within about 2 weeksafter initiation of treatment, and to maintain the plasma trough levelof at least about 1,000 ng/mL of the HhP inhibitor for the duration ofthe treatment.
 9. The method of claim 1, further comprising measuringthe plasma level of the HhP inhibitor, or a metabolite thereof, in thesubject one or more times.
 10. The method of claim 9, wherein saidmeasuring is carried out one or more times about 4 weeks afterinitiation of treatment with the HhP inhibitor.
 11. The method of claim1, further comprising measuring the plasma level of the HhP inhibitor,or a metabolite thereof, one or more times in a period of time fromabout 4 weeks to about 12 weeks.
 12. The method of claim 11, furthercomprising increasing a subsequent dose of the HhP inhibitor if theplasma trough level of at least about 1,000 ng/mL of the HhP inhibitoris not maintained.
 13. The method of claim 11, further comprisingreducing a subsequent dose of an HhP inhibitor if the plasma troughlevel at about 4 weeks is at least 1000 ng/mL and the patient isexperiencing one or more side effects.
 14. The method of claim 1,wherein the proliferation disorder is cancer.
 15. The method of claim 1,wherein the proliferation disorder is prostate cancer and said methodfurther comprises comparing the level of prostate-specific antigen (PSA)in a sample obtained from the subject following administration of theHhP inhibitor with a reference level of PSA, and wherein the level ofPSA in the sample compared to the reference level of PSA is prognosticfor an outcome of treatment with the HhP inhibitor.
 16. The method ofclaim 1, further comprising monitoring the proliferation disorder in thesubject, wherein a lack of clinical response in the proliferationdisorder to the treatment is indicative that the plasma trough level ofthe HhP inhibitor should be increased further above about 1000 ng/mL,and wherein the occurrence of a clinical response and a plasma troughlevel of the HhP inhibitor substantially higher than about 1000 ng/mLindicates that one or more subsequent doses of the HhP inhibitor can bereduced.
 17. The method of claim 1, further comprising monitoring theproliferation disorder in the subject, wherein a lack of clinicalresponse in the proliferation disorder to the treatment, after aboutfour weeks of said administering, is indicative of a need to increasethe dose of the HhP inhibitor, or increase the frequency of the dose ofthe HhP inhibitor, or administer an additional HhP inhibitor thatinhibits the HhP by a different mechanism than the previouslyadministered HhP inhibitor, or a combination of two or more of theforegoing.
 18. The method of claim 1, wherein the proliferation disorderis prostate cancer, wherein the composition comprises a SUBA®formulation of itraconazole, or a pharmaceutically acceptable salt,prodrug, stereoisomer, or active metabolite thereof, and wherein theSUBA® formulation is orally administered at a dose in the range of 100mg to 600 mg per day.
 19. The method of claim 1, wherein theproliferation disorder is basal cell carcinoma, wherein the compositioncomprises a SUBA® formulation of itraconazole, or a pharmaceuticallyacceptable salt, prodrug, stereoisomer, or active metabolite thereof,and wherein the SUBA® formulation is orally administered at a dose inthe range of 100 mg to 600 mg per day.
 20. The method of claim 1,wherein the proliferation disorder is lung cancer, wherein thecomposition comprises a SUBA® formulation of itraconazole, or apharmaceutically acceptable salt, prodrug, stereoisomer, or activemetabolite thereof, wherein the SUBA® formulation is orally administeredat a dose in the range of 100 mg to 600 mg per day, and wherein themethod further comprises administering an antifolate and aplatinum-based chemotherapeutic agent to the subject.